Combination therapy using t cell redirection antigen binding molecule against cell having immunosuppressing function

ABSTRACT

It was discovered that antigen-binding molecules comprising (1) a domain that binds to a molecule expressed on the surface of cells having immune response-suppressing functions and (2) a T cell receptor complex-binding domain, crosslink T cells with the cells having immune response-suppressing functions and induce damage to the cells having immune response-suppressing functions to thereby exhibit more superior antitumor effects than conventional antigen-binding molecules. It was also discovered that combined use of the antigen-binding molecules and other anticancer agents further increases the antitumor effects.

TECHNICAL FIELD

The present invention relates to T cell-redirecting antigen-bindingmolecules that enable the treatment of various types of cancers bydamaging cells having an immune response-suppressing function; andcombination therapies that use the T cell-redirecting antigen-bindingmolecules.

BACKGROUND ART

Antibodies are drawing attention as pharmaceuticals since they arehighly stable in plasma and have few side effects. In particular, anumber of IgG-type antibody pharmaceuticals are available on the marketand many antibody pharmaceuticals are currently being developed(Non-patent Documents 1 and 2).

As cancer therapeutic agents using antibody pharmaceuticals, Rituxanagainst the CD20 antigen, Cetuximab against the EGFR antigen, Herceptinagainst the HER2 antigen, and such have been approved so far (Non-patentDocument 3). These antibody molecules bind to antigens expressed oncancer cells, and exhibit cytotoxic activity against cancer cellsthrough ADCC and such.

Apart from such antibody pharmaceuticals that exhibit cytotoxicactivities directly against cancer cells, Ipilimumab which is anIgG1-type antibody against CTLA4 was recently confirmed to be effectiveagainst metastatic melanoma, and received FDA approval in 2011. In solidcancer microenvironments, effector T cells that recognize tumors areknown to be suppressed by various factors in the cancer microenvironment(Non-patent Document 4). Among them, CTLA4 which is an immune checkpointmolecule is known to have the function of suppressing T cell activation,and is strongly expressed in activated effector T cells and regulatory Tcells. In cancer microenvironments, tumor-recognizing effector T cellsare considered to be suppressed by CTLA4. Ipilimumab is a neutralizingantibody against CTLA4, and it is considered to exhibit antitumoreffects by inhibiting the suppression of effector T cell-activation andactivating the tumor-recognizing effector T cells (Non-patent Document5). Similarly, nivolumab which is a neutralizing antibody against theimmune checkpoint molecule PD1 has been recently reported to be alsohighly effective against metastatic melanoma by the activation oftumor-recognizing effector T cells that are suppressed by PD1(Non-patent Document 6).

Recently, clinical trials of combined use of -antibody drugs blockingdifferent immune checkpoint molecules have been performed on varioustumors (Non-patent Document 7), and combined use of an IgG1-typeanti-CTLA4 antibody, Ipilimumab and an anti-PD1 antibody against theimmune checkpoint molecule PD1 have been tested in clinical trialstargeting multiple types of cancers (Non-patent Documents 8 and 9).Furthermore, in experiments using tumor-bearing mouse models,synergistic anti-tumor effects have been confirmed when administering ananti-CTLA4 antibody in combination with a plurality of chemotherapeuticagents (Non-patent Document 10).

Furthermore, not only regulatory T cells but also exhausted T cells areknown to be present in the cancer microenvironment, and the exhausted Tcells are known to not only lack cytotoxic activity (antitumor activity)but also to contribute on immunosuppression in tumors (Non-patentDocument 11).

Thus, it was thought that antibody pharmaceuticals against immunecheckpoint molecules exert antitumor effects by inhibitingT-cell-suppressing effects of immune checkpoint molecules. However, ithas been recently reported that for exhibiting antitumor effects ofanti-CTLA4 antibodies such as ipilimumab, not only the neutralizingeffects on CTLA4, but the antibody-dependent cytotoxic activity (ADCCactivity) against CTLA4-expressing cells is also important (Non-patentDocuments 12 and 13). Regulatory T cells and exhausted T cells in thetumor microenvironment are known to strongly express CTLA4, andanti-CTLA4 antibodies having ADCC activity are thought to be able tokill the regulatory T cells and exhausted T cells. In Non-patentDocuments 12 and 13, while anti-CTLA4 antibodies having ADCC activitycould eliminate regulatory T cells in tumors and exert strong antitumoreffects in mice, anti-CTLA4 antibodies whose ADCC activity has beenremoved by amino acid substitutions could not eliminate regulatory Tcells in tumors and could not exert antitumor effects.

Thus, it has been found that ADCC activity plays an important role inantitumor effects of anti-CTLA4 antibodies, and that the killing ofregulatory T cells by ADCC activity is an important mechanism of action.

On the other hand, ADCC activity of an antibody is exhibited by bindingof its Fc region to an Fcγ receptor. For example, substituting(modifying) amino acids of the Fc region of an IgG1 antibody to increasebinding to the Fcγ receptor has been reported to enable enhancement ofADCC activity (Non-Patent Document 14). In fact, an anti-CD19 antibodywith enhanced ADCC activity due to modification of the Fc region exertedstronger antitumor effects than natural anti-CD19 antibodies (Non-patentDocument 14).

PRIOR ART DOCUMENTS

[Non-Patent Documents]

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Cancer Med. 2013 October;    2(5):662-73.-   [Non-patent Document 7] Combined Immune Checkpoint Blockade.    Charles G. Drake. Semin Oncol. 2015 August; 42(4):656-62-   [Non-patent Document 8] Reorienting the immune system in the    treatment of cancer by using anti-PD-1 and anti-PD-L1 antibodies.    Troels H. Borch, Marco Donia, Mads H. Andersen, Inge M. Svane. Drug    Discov Today. 2015 September; 20(9): 1127-34-   [Non-patent Document 9] Nivolumab and ipilimumab versus ipilimumab    in untreated melanoma. Postow M A, Chesney J, Pavlick A C, Robert C,    Grossmann K, McDermott D, Linette G P, Meyer N, Giguere J K,    Agarwala S S, Shaheen M, Emstoff M S, Minor D, Salama A K, Taylor M,    Ott P A, Rollin L M, Horak C, Gagnier P, Wolchok J D, Hodi F S. N    Engl J Med. 2015 May 21; 372(21):2006-17-   [Non-patent Document 10] Synergy between chemotherapeutic agents and    CTLA-4 blockade in preclinical tumor models. 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Korman, Cancer Immunol Res; 1(1); 1-11.-   [Non-patent Document 14] The impact of Fc engineering on an    anti-CD19 antibody: increased Fcgamma receptor affinity enhances    B-cell clearing in nonhuman primates. Zalevsky J, Leung I W, Karki    S, Chu S Y, Zhukovsky E A, Desjarlais J R, Carmichael D F, Lawrence    C E. Blood. 2009 Apr. 16; 113(16):3735-43.-   [Non-patent Document 15] Curiel, T. J. et al. Specific recruitment    of regulatory T cells in ovarian carcinoma fosters immune privilege    and predicts reduced survival. Nature medicine 10, 942-949, doi:    10.1038/nm1093 (2004).-   [Non-patent Document 16] Sato, E. et al. Intraepithelial CD8⁺    tumor-infiltrating lymphocytes and a high CD8/regulatory T cell    ratio are associated with favorable prognosis in ovarian cancer.    Proceedings of the National Academy of Sciences of the United States    of America 102, 18538-18543, doi:10.1073/pnas.0509182102 (2005).-   [Non-patent Document 17] Kawaida, H. et al. 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Quantification of    regulatory T cells enables the identification of high-risk breast    cancer patients and those at risk of late relapse. Journal of    clinical oncology: official journal of the American Society of    Clinical Oncology 24, 5373-5380, doi: 10.1200/JCO.2006.05.9584    (2006).-   [Non-patent Document 24] Gerber, A. L. et al. High expression of    FOXP3 in primary melanoma is associated with tumour progression. The    British journal of dermatology 170, 103-109, doi:10.1111/bjd.12641    (2014).-   [Non-patent Document 25] Wang, Y. Y., He, X. Y., Cai, Y. Y.,    Wang, Z. J. & Lu, S. H. The variation of CD4⁺CD25⁺ regulatory T    cells in the periphery blood and tumor microenvironment of non-small    cell lung cancer patients and the downregulation effects induced by    CpG ODN. Targeted oncology 6, 147-154, doi:    10.1007/s11523-011-0182-9 (2011).-   [Non-patent Document 26] de Vos van Steenwijk, P. J. et al.    Tumor-infiltrating CD14-positive myeloid cells and CD8-positive    T-cells prolong survival in patients with cervical carcinoma.    International journal of cancer. Journal international du cancer    133, 2884-2894, doi: 10.1002/ijc.28309 (2013).-   [Non-patent Document 27] Wainwright, D. A., Dey, M., Chang, A. &    Lesniak, M. S. Targeting Tregs in Malignant Brain Cancer: Overcoming    IDO. Frontiers in immunology 4, 116, doi:10.3389/fimmu.2013.00116    (2013).-   [Non-patent Document 28] Yu, P. et al. Simultaneous inhibition of    two regulatory T-cell subsets enhanced Interleukin-15 efficacy in a    prostate tumor model. Proceedings of the National Academy of    Sciences of the United States of America 109, 6187-6192, doi:    10.1073/pnas. 1203479109 (2012).-   [Non-patent Document 29] Zheng, J., Liu, P. & Yang, X. YB-1    immunization combined with regulatory T-cell depletion induces    specific T-cell responses that protect against neuroblastoma in the    early stage. 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E. & Ansell, S. M. Attenuation of CD8⁺ T-cell function by    CD4⁺CD25⁺ regulatory T cells in B-cell non-Hodgkin's lymphoma.    Cancer research 66, 10145-10152, doi: 10.1158/0008-5472.CAN-06-1822    (2006).-   [Non-patent Document 34] Yang, Z. Z., Novak, A. J., Stenson, M. J.,    Witzig, T. E. & Ansell, S. M. Intratumoral CD4⁺CD25⁺ regulatory    T-cell-mediated suppression of infiltrating CD4⁺ T cells in B-cell    non-Hodgkin lymphoma. Blood 107, 3639-3646, doi:    10.1182/blood-2005-08-3376 (2006).

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.The present invention provides T cell-redirecting antigen-bindingmolecules that exhibit excellent antitumor effects by damaging cellshaving an immune response-suppressing function; and combinationtherapies that use the T cell-redirecting antigen-binding molecules.

Means for Solving the Problems

The present inventors discovered that T cell-redirecting antigen-bindingmolecules comprising a domain that binds to a molecule expressed on thesurface of cells having immune response-suppressing functions and adomain that binds to a T-cell receptor (TCR) complex, damage the cellshaving immune response-suppressing functions and exhibit more superiorantitumor activity than conventional T cell-redirecting antigen-bindingmolecules that bind to molecules expressed on the surfaces of regulatoryT cells and exhausted T cells and that also have ADCC activity.Furthermore, the present inventors discovered methods for treating orpreventing cancer by combined use of the T cell-redirectingantigen-binding molecule and an anticancer agent; and T cell-redirectingantigen-binding molecules, anticancer agents, or pharmaceuticalcompositions comprising a combination of a T cell-redirectingantigen-binding molecule and an anticancer agent, each of which is usedin the combination therapies.

More specifically, the present invention provides the following:

-   [1] a pharmaceutical composition comprising as an active ingredient    a first antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function,        -   wherein the pharmaceutical composition is for use in            combination with an anticancer agent;-   [2] the pharmaceutical composition of [1], wherein the first    antigen-binding molecule is administered simultaneously with the    anticancer agent;-   [3] the pharmaceutical composition of [1], wherein the first    antigen-binding molecule is administered before or after    administration of the anticancer agent;-   [4] the pharmaceutical composition of any one of [1] to [3], wherein    the first antigen-binding molecule further comprises an FcRn-binding    domain;-   [5] the pharmaceutical composition of [4], wherein the FcRn-binding    domain is an antibody Fc region having decreased Fcγ    receptor-binding activity;-   [6] the pharmaceutical composition of any one of [1] to [5], wherein    the first antigen-binding molecule is a multispecific antibody;-   [7] the pharmaceutical composition of any one of [1] to [6], wherein    the cell having an immune response-suppressing function is a    regulatory T cell or an exhausted T cell;-   [8] the pharmaceutical composition of any one of [1] to [7], wherein    the molecule expressed on the surface of the cell having an immune    response-suppressing function is any molecule selected from among    CTLA4, PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137    (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6,    CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20,    CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b,    Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT;-   [9] the pharmaceutical composition of any one of [1] to [8], wherein    the T cell receptor complex-binding domain is a T cell    receptor-binding domain or a CD3-binding domain;-   [10] the pharmaceutical composition of any one of [1] to [9],    wherein the anticancer agent is an alkylating agent, an    antimetabolite, a plant alkaloid, an antibiotic, a platinum    compound, methylhydrazine, a kinase inhibitor, an enzyme, a histone    deacetylase inhibitor, a retinoid, an antibody, or an immune    checkpoint inhibitor;-   [11] the pharmaceutical composition of any one of [1] to [9],    wherein the anticancer agent is a second antigen-binding molecule    comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a tumor necrosis factor (TNF) superfamily-binding domain or        tumor necrosis factor (TNF) receptor superfamily-binding domain;-   [12] the pharmaceutical composition of any one of [1] to [9],    wherein the anticancer agent is a third antigen-binding molecule    comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a T cell receptor complex-binding domain;-   [13] the pharmaceutical composition of [11] or [12], wherein the    second antigen-binding molecule and/or the third antigen-binding    molecule further comprises an FcRn-binding domain;-   [14] the pharmaceutical composition of [13], wherein the    FcRn-binding domain of the second antigen-binding molecule and/or    the third antigen-binding molecule is an antibody Fc region having    decreased Fcγ receptor-binding activity;-   [15] the pharmaceutical composition of any one of [11] to [14],    wherein the second antigen-binding molecule and/or the third    antigen-binding molecule is a bispecific antibody;-   [16] the pharmaceutical composition of any one of [11] to [15],    wherein the cancer-specific antigen-binding domain of the second    antigen-binding molecule and/or the third antigen-binding molecule    is a GPC3-binding domain;-   [17] the pharmaceutical composition of any one of [11] and [13] to    [16], wherein the TNF superfamily-binding domain or the TNF receptor    superfamily-binding domain of the second antigen-binding molecule is    a CD137-binding domain or a CD40-binding domain;-   [18] the pharmaceutical composition of any one of [12] to [17],    wherein the T cell receptor complex-binding domain of the third    antigen-binding molecule is a T cell receptor-binding domain or a    CD3-binding domain;-   [19] the pharmaceutical composition of any one of [1] to [18], which    is a pharmaceutical composition for treating or preventing any    cancer selected from the group consisting of ovarian cancer, gastric    cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma,    hepatocellular carcinoma, breast cancer, malignant melanoma,    non-small-cell lung cancer, cervical cancer, glioblastoma, prostate    cancer, neuroblastoma, chronic lymphocytic leukemia, papillary    thyroid cancer, colorectal cancer, and B-cell non-Hodgkin's    lymphoma; and-   [20] an agent for inducing cytotoxicity, an agent for suppressing    cell proliferation, an agent for inhibiting cell proliferation, an    agent for activating an immune response, an agent for treating    cancer, or an agent for preventing cancer, each of which comprises    the pharmaceutical composition of any one of [1] to [19].

Furthermore, the following inventions are also provided:

-   [21] a pharmaceutical composition comprising an anticancer agent as    an active ingredient, wherein the pharmaceutical composition is for    use in combination with a first antigen-binding molecule that    comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [22] the pharmaceutical composition of [21], wherein the anticancer    agent is administered simultaneously with the first antigen-binding    molecule;-   [23] the pharmaceutical composition of [21], wherein the anticancer    agent is administered before or after administration of the first    antigen-binding molecule;-   [24] a pharmaceutical composition for treating or preventing cancer,    the composition comprising a combination of an anticancer agent and    a first antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [25] the pharmaceutical composition of [24], which is a combination    preparation;-   [26] the pharmaceutical composition of [24], wherein the first    antigen-binding molecule and the anticancer agent are administered    separately;-   [27] the pharmaceutical composition of [26], wherein the first    antigen-binding molecule and the anticancer agent are administered    simultaneously or sequentially;-   [28] the pharmaceutical composition of any one of [21] to [27],    wherein the first antigen-binding molecule further comprises an    FcRn-binding domain;-   [29] the pharmaceutical composition of [28] wherein the FcRn-binding    domain is an antibody Fc region having decreased Fcγ    receptor-binding activity;-   [30] the pharmaceutical composition of any one of [21] to [29],    wherein the first antigen-binding molecule is a multispecific    antibody;-   [31] the pharmaceutical composition of any one of [21] to [30],    wherein the cell having an immune response-suppressing function is a    regulatory T cell or an exhausted T cell;-   [32] the pharmaceutical composition of any one of [21] to [31],    wherein the molecule expressed on the surface of the cell having an    immune response-suppressing function is any molecule selected from    among CTLA4, PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137    (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6,    CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20,    CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b,    Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT;-   [33] the pharmaceutical composition of any one of [21] to [32],    wherein the T cell receptor complex-binding domain is a T cell    receptor-binding domain or a CD3-binding domain;-   [34] the pharmaceutical composition of any one of [21] to [33],    wherein the anticancer agent is an alkylating agent, an    antimetabolite, a plant alkaloid, an antibiotic, a platinum    compound, methylhydrazine, a kinase inhibitor, an enzyme, a histone    deacetylase inhibitor, a retinoid, an antibody, or an immune    checkpoint inhibitor;-   [35] the pharmaceutical composition of any one of [21] to [34],    wherein the anticancer agent is a second antigen-binding molecule    comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a tumor necrosis factor (TNF) superfamily-binding domain or        a tumor necrosis factor (TNF) receptor superfamily-binding        domain;-   [36] the pharmaceutical composition of any one of [21] to [35],    wherein the anticancer agent is a third antigen-binding molecule    comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a T cell receptor complex-binding domain;-   [37] the pharmaceutical composition of [35] or [36], wherein the    second antigen-binding molecule and/or the third antigen-binding    molecule further comprises an FcRn-binding domain;-   [38] the pharmaceutical composition of [37], wherein the    FcRn-binding domain of the second antigen-binding molecule and/or    the third antigen-binding molecule is an antibody Fc region having    decreased Fcγ receptor-binding activity;-   [39] the pharmaceutical composition of any one of [35] to [38],    wherein the second antigen-binding molecule and/or the third    antigen-binding molecule is a bispecific antibody;-   [40] the pharmaceutical composition of any one of [35] to [39],    wherein the cancer-specific antigen-binding domain of the second    antigen-binding molecule and/or the third antigen-binding molecule    is a GPC3-binding domain;-   [41] the pharmaceutical composition of any one of [35] and [37] to    [40], wherein the TNF superfamily-binding domain or the TNF receptor    superfamily-binding domain of the second antigen-binding molecule is    a CD137-binding domain or a CD40-binding domain;-   [42] the pharmaceutical composition of any one of [36] to [41],    wherein the T cell receptor complex-binding domain of the third    antigen-binding molecule is a T cell receptor-binding domain or a    CD3-binding domain;-   [43] the pharmaceutical composition of any one of [21] to [42],    which is a pharmaceutical composition for treating or preventing any    cancer selected from the group consisting of ovarian cancer, gastric    cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma,    hepatocellular carcinoma, breast cancer, malignant melanoma,    non-small-cell lung cancer, cervical cancer, glioblastoma, prostate    cancer, neuroblastoma, chronic lymphocytic leukemia, papillary    thyroid cancer, colorectal cancer, and B-cell non-Hodgkin's    lymphoma; and-   [44] an agent for inducing cytotoxicity, an agent for suppressing    cell proliferation, an agent for inhibiting cell proliferation, an    agent for activating immune response, an agent for treating cancer,    or an agent for preventing cancer, each of which comprises the    pharmaceutical composition of any one of [21] to [43].

Furthermore, the following inventions are also provided:

-   [45] a combination of an anticancer agent and a first    antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [46] the combination according to [45], wherein the first    antigen-binding molecule is administered simultaneously with the    anticancer agent;-   [47] the combination according to [45], wherein the first    antigen-binding molecule is administered before or after    administration of the anticancer agent;-   [48] the combination according to any one of [45] to [47], wherein    the first antigen-binding molecule further comprises an FcRn-binding    domain;-   [49] the combination according to [48] wherein the FcRn-binding    domain is an antibody Fc region having decreased Fcγ    receptor-binding activity;-   [50] the combination according to any one of [45] to [49], wherein    the first antigen-binding molecule is a multispecific antibody;-   [51] the combination according to any one of [45] to [50], wherein    the cell having an immune response-suppressing function is a    regulatory T cell or an exhausted T cell;-   [52] the combination according to any one of [45] to [51], wherein    the molecule expressed on the surface of the cell having an immune    response-suppressing function is any molecule selected from among    CTLA4, PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137    (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6,    CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20,    CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b,    Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT;-   [53] the combination according to any one of [45] to [52], wherein    the T cell receptor complex-binding domain is a T cell    receptor-binding domain or a CD3-binding domain;-   [54] the combination according to any one of [45] to [53], wherein    the anticancer agent is an alkylating agent, an antimetabolite, a    plant alkaloid, an antibiotic, a platinum compound, methylhydrazine,    a kinase inhibitor, an enzyme, a histone deacetylase inhibitor, a    retinoid, an antibody, or an immune checkpoint inhibitor;-   [55] the combination according to any one of [45] to [53], wherein    the anticancer agent is a second antigen-binding molecule    comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a tumor necrosis factor (TNF) superfamily-binding domain or        tumor necrosis factor (TNF) receptor superfamily-binding domain;-   [56] the combination according to any one of [45] to [53], wherein    the anticancer agent is a third antigen-binding molecule comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a T cell receptor complex-binding domain;-   [57] the combination according to [55] or [56], wherein the second    antigen-binding molecule and/or the third antigen-binding molecule    further comprises an FcRn-binding domain;-   [58] the combination according to [57], wherein the FcRn-binding    domain of the second antigen-binding molecule and/or the third    antigen-binding molecule is an antibody Fc region having decreased    Fcγ receptor-binding activity;-   [59] the combination according to any one of [55] to [58], wherein    the second antigen-binding molecule and/or the third antigen-binding    molecule is a bispecific antibody;-   [60] the combination according to any one of [55] to [59], wherein    the cancer-specific antigen-binding domain of the second    antigen-binding molecule and/or the third antigen-binding molecule    is a GPC3-binding domain;-   [61] the combination according to any one of [55] and [57] to [60],    wherein the TNF superfamily-binding domain or the TNF receptor    superfamily-binding domain of the second antigen-binding molecule is    a CD137-binding domain or a CD40-binding domain;-   [62] the combination according to any one of [56] to [61], wherein    the T cell receptor complex-binding domain of the third    antigen-binding molecule is a T cell receptor-binding domain or a    CD3-binding domain;-   [63] the combination according to any one of [45] to [62], which is    for treating or preventing any cancer selected from the group    consisting of ovarian cancer, gastric cancer, esophageal cancer,    pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma,    breast cancer, malignant melanoma, non-small-cell lung cancer,    cervical cancer, glioblastoma, prostate cancer, neuroblastoma,    chronic lymphocytic leukemia, papillary thyroid cancer, colorectal    cancer, and B-cell non-Hodgkin's lymphoma; and-   [64] an agent for inducing cytotoxicity, an agent for suppressing    cell proliferation, an agent for inhibiting cell proliferation, an    agent for activating immune response, an agent for treating cancer,    or an agent for preventing cancer, each of which comprises the    combination according to any one of [45] to [63].

Furthermore, the following inventions are also provided:

-   [65] a method for inducing cytotoxicity, for suppressing cell    proliferation, for inhibiting cell proliferation, for activating    immune response, for treating cancer, or for preventing cancer in an    individual, each method comprising administering an effective amount    of an anticancer agent and an effective amount of a first    antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [66] a method for inducing cytotoxicity, for suppressing cell    proliferation, for inhibiting cell proliferation, for activating    immune response, for treating cancer, or for preventing cancer in an    individual, each method comprising administering an effective amount    of an anticancer agent to the individual with combined use of a    first antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [67] a method for inducing cytotoxicity, for suppressing cell    proliferation, for inhibiting cell proliferation, for activating    immune response, for treating cancer, or for preventing cancer in an    individual, each method comprising administering to an individual,    with combined use of an anticancer agent, an effective amount of a    first antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [68] a method for enhancing effects of inducing cytotoxicity,    suppressing cell proliferation, inhibiting cell proliferation,    activating immune response, treating cancer, or preventing cancer in    an individual by a first antigen-binding molecule, each method    comprising administering an effective amount of an anticancer agent    to an individual, wherein the first antigen-binding molecule    comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [69] a method for enhancing effects of inducing cytotoxicity,    suppressing cell proliferation, inhibiting cell proliferation,    activating immune response, treating cancer, or preventing cancer in    an individual by an anticancer agent, each method comprising    administering to an individual an effective amount of a first    antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [70] the method of any one of [29] to [33], wherein the first    antigen-binding molecule and the anticancer agent are administered    separately;-   [71] the method of any one of [29] to [33], wherein the first    antigen-binding molecule and the anticancer agent are administered    simultaneously or sequentially;-   [72] the method of any one of [65] to [71], wherein the first    antigen-binding molecule further comprises an FcRn-binding domain;-   [73] the method of [72], wherein the FcRn-binding domain is an    antibody Fc region having decreased Fcγ receptor-binding activity;-   [74] the method of any one of [65] to [73], wherein the first    antigen-binding molecule is a multispecific antibody;-   [75] the method of any one of [65] to [74], wherein the cell having    an immune response-suppressing function is a regulatory T cell or an    exhausted T cell;-   [76] the method of any one of [64] to [75], wherein the molecule    expressed on the surface of the cell having an immune    response-suppressing function is any molecule selected from among    CTLA4, PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137    (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6,    CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20,    CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b,    Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT;-   [77] the method of any one of [65] to [76], wherein the T cell    receptor complex-binding domain is a T cell receptor-binding domain    or a CD3-binding domain;-   [78] the method of any one of [65] to [77], wherein the anticancer    agent is an alkylating agent, an antimetabolite, a plant alkaloid,    an antibiotic, a platinum compound, methylhydrazine, a kinase    inhibitor, an enzyme, a histone deacetylase inhibitor, a retinoid,    an antibody, or an immune checkpoint inhibitor;-   [79] the method of any one of [65] to [77], wherein the anticancer    agent is a second antigen-binding molecule comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a tumor necrosis factor (TNF) superfamily-binding domain or        a tumor necrosis factor (TNF) receptor superfamily-binding        domain;-   [80] the method of any one of [65] to [77], wherein the anticancer    agent is a third antigen-binding molecule comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a T cell receptor complex-binding domain;-   [81] the method of [79] or [80], wherein the second antigen-binding    molecule and/or the third antigen-binding molecule further comprises    an FcRn-binding domain;-   [82] the method of [81], wherein the FcRn-binding domain of the    second antigen-binding molecule and/or the third antigen-binding    molecule is an antibody Fc region having decreased Fcγ    receptor-binding activity;-   [83] the method of any one of [79] to [82], wherein the second    antigen-binding molecule and/or the third antigen-binding molecule    is a bispecific antibody;-   [84] the method of any one of [79] to [83], wherein the    cancer-specific antigen-binding domain of the second antigen-binding    molecule and/or the third antigen-binding molecule is a GPC3-binding    domain;-   [85] the method of any one of [79] and [81] to [84], wherein the TNF    superfamily-binding domain or the TNF receptor superfamily-binding    domain of the second antigen-binding molecule is a CD137-binding    domain or a CD40-binding domain;-   [86] the method of any one of [80] to [85], wherein the T cell    receptor complex-binding domain of the third antigen-binding    molecule is a T cell receptor-binding domain or a CD3-binding    domain; and-   [87] the method of any one of [65] to [86], wherein the cancer is    any cancer selected from the group consisting of ovarian cancer,    gastric cancer, esophageal cancer, pancreatic cancer, renal cell    carcinoma, hepatocellular carcinoma, breast cancer, malignant    melanoma, non-small-cell lung cancer, cervical cancer, glioblastoma,    prostate cancer, neuroblastoma, chronic lymphocytic leukemia,    papillary thyroid cancer, colorectal cancer, and B-cell    non-Hodgkin's lymphoma.

Furthermore, the following inventions are also provided:

-   [88] a kit comprising:    -   (A) a pharmaceutical composition comprising a first        antigen-binding molecule that comprises:        -   (1) a domain that binds to a molecule expressed on the            surface of a cell having an immune response-suppressing            function; and        -   (2) a T cell receptor complex-binding domain, and        -   inhibits the immune response-suppressing activity of the            cell having an immune response-suppressing function;    -   (B) a container; and    -   (C) an instruction or a label indicating that the first        antigen-binding molecule and at least one type of anticancer        agent are administered in combination to an individual for        treating or preventing cancer in the individual;-   [89] a kit comprising:    -   (A) an anticancer agent;    -   (B) a container; and    -   (C) an instruction or a label indicating that the anticancer        agent and a pharmaceutical composition comprising at least one        type of a first antigen-binding molecule are administered in        combination to an individual for treating or preventing cancer        in the individual, wherein the first antigen-binding molecule        comprises:        -   (1) a domain that binds to a molecule expressed on the            surface of a cell having an immune response-suppressing            function; and        -   (2) a T cell receptor complex-binding domain, and        -   inhibits the immune response-suppressing activity of the            cell having an immune response-suppressing function;-   [90] a kit comprising:    -   (A) a pharmaceutical composition comprising a first        antigen-binding molecule that comprises:        -   (1) a domain that binds to a molecule expressed on the            surface of a cell having an immune response-suppressing            function; and        -   (2) a T cell receptor complex-binding domain, and        -   inhibits the immune response-suppressing activity of the            cell having an immune response-suppressing function;    -   (B) a container; and    -   (C) an anticancer agent;-   [91] the kit of [88], wherein the first antigen-binding molecule and    the anticancer agent are administered simultaneously;-   [92] the kit of [88], wherein the first antigen-binding molecule is    administered before or after administration of the anticancer agent;-   [93] the kit of any one of [88] to [92], wherein the first    antigen-binding molecule further comprises an FcRn-binding domain;-   [94] the kit of [93], wherein the FcRn-binding domain is an antibody    Fc region having decreased Fcγ receptor-binding activity;-   [95] the kit of any one of [88] to [94], wherein the first    antigen-binding molecule is a multispecific antibody;-   [96] the kit of any one of [88] to [95], wherein the cell having an    immune response-suppressing function is a regulatory T cell or an    exhausted T cell;-   [97] the kit of any one of [88] to [96], wherein the molecule    expressed on the surface of the cell having an immune    response-suppressing function is any molecule selected from among    CTLA4, PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137    (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6,    CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20,    CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b,    Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT;-   [98] the kit of any one of [88] to [97], wherein the T cell receptor    complex-binding domain is a T cell receptor-binding domain or a    CD3-binding domain;-   [99] the kit of any one of [88] to [98], wherein the anticancer    agent is an alkylating agent, an antimetabolite, a plant alkaloid,    an antibiotic, a platinum compound, methylhydrazine, a kinase    inhibitor, an enzyme, a histone deacetylase inhibitor, a retinoid,    an antibody, or an immune checkpoint inhibitor;-   [100] the kit of any one of [88] to [98], wherein the anticancer    agent is a second antigen-binding molecule comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a tumor necrosis factor (TNF) superfamily-binding domain or        a tumor necrosis factor (TNF) receptor superfamily-binding        domain;-   [101] the kit of any one of [88] to [98], wherein the anticancer    agent is a third antigen-binding molecule comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a T cell receptor complex-binding domain;-   [102] the kit of [100] or [101], wherein the second antigen-binding    molecule and/or the third antigen-binding molecule further comprises    an FcRn-binding domain;-   [103] the kit of [102], wherein the FcRn-binding domain of the    second antigen-binding molecule and/or the third antigen-binding    molecule is an antibody Fc region having decreased Fcγ    receptor-binding activity;-   [104] the kit of any one of [100] to [103], wherein the second    antigen-binding molecule and/or the third antigen-binding molecule    is a bispecific antibody;-   [105] the kit of any one of [100] to [104], wherein the    cancer-specific antigen-binding domain of the second antigen-binding    molecule and/or the third antigen-binding molecule is a GPC3-binding    domain;-   [106] the kit of any one of [100] and [102] to [105], wherein the    TNF superfamily-binding domain or the TNF receptor    superfamily-binding domain of the second antigen-binding molecule is    a CD137-binding domain or a CD40-binding domain;-   [107] the kit of any one of [101] to [106], wherein the T cell    receptor complex-binding domain of the third antigen-binding    molecule is a T cell receptor-binding domain or a CD3-binding    domain; and-   [108] the kit of any one of [101] to [107], wherein the cancer is    any cancer selected from the group consisting of ovarian cancer,    gastric cancer, esophageal cancer, pancreatic cancer, renal cell    carcinoma, hepatocellular carcinoma, breast cancer, malignant    melanoma, non-small-cell lung cancer, cervical cancer, glioblastoma,    prostate cancer, neuroblastoma, chronic lymphocytic leukemia,    papillary thyroid cancer, colorectal cancer, and B-cell    non-Hodgkin's lymphoma.

Furthermore, the following inventions are also provided:

-   [109] a method for inducing damage to a cancer cell or a cancer    cell-comprising tumor tissue, or a method for suppressing    proliferation of a cancer cell or a cancer cell-comprising tumor    tissue, by contacting a cell having an immune response-suppressing    function and a cancer cell with an anticancer agent and a first    antigen-binding molecule that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of the cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [110] a method for assessing whether a first antigen-binding    molecule and an anticancer agent induce damage to a cancer cell or a    cancer cell-comprising tumor tissue, or suppress proliferation of a    cancer cell or a cancer cell-comprising tumor tissue, by contacting    a cell having an immune response-suppressing function and a cancer    cell with an anticancer agent and a first antigen-binding molecule    that comprises:    -   (1) a domain that binds to a molecule expressed on the surface        of the cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and    -   inhibits the immune response-suppressing activity of the cell        having an immune response-suppressing function;-   [111] the method of [109] or [110], wherein the first    antigen-binding molecule and the anticancer agent are simultaneously    contacted with the cell having an immune response-suppressing    function and the cancer cell;-   [112] the method of [109] or [110], wherein the first    antigen-binding molecule is contacted with the cell having an immune    response-suppressing function and the cancer cell before or after    the anticancer agent;-   [113] the method of any one of [109] to [112], wherein the first    antigen-binding molecule further comprises an FcRn-binding domain;-   [114] the method of [113], wherein the FcRn-binding domain is an    antibody Fc region having decreased Fcγ receptor-binding activity;-   [115] the method of any one of [109] to [114], wherein the first    antigen-binding molecule is a multispecific antibody;-   [116] the method of any one of [109] to [115], wherein the cell    having an immune response-suppressing function is a regulatory T    cell or an exhausted T cell;-   [117] the method of any one of [109] to [116], wherein the molecule    expressed on the surface of the cell having an immune    response-suppressing function is any molecule selected from among    CTLA4, PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137    (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6,    CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20,    CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b,    Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT;-   [118] the method of any one of [109] to [117], wherein the T cell    receptor complex-binding domain is a T cell receptor-binding domain    or a CD3-binding domain;-   [119] the method of any one of [109] to [118], wherein the    anticancer agent is an alkylating agent, an antimetabolite, a plant    alkaloid, an antibiotic, a platinum compound, methylhydrazine, a    kinase inhibitor, an enzyme, a histone deacetylase inhibitor, a    retinoid, an antibody, or an immune checkpoint inhibitor;-   [120] the method of any one of [109] to [118], wherein the    anticancer agent is a second antigen-binding molecule comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a tumor necrosis factor (TNF) superfamily-binding domain or        a tumor necrosis factor (TNF) receptor superfamily-binding        domain;-   [121] the method of any one of [109] to [118], wherein the    anticancer agent is a third antigen-binding molecule comprising:    -   (1) a cancer-specific antigen-binding domain; and    -   (2) a T cell receptor complex-binding domain;-   [122] the method of [120] or [121], wherein the second    antigen-binding molecule and/or the third antigen-binding molecule    further comprises an FcRn-binding domain;-   [123] the method of [122], wherein the FcRn-binding domain of the    second antigen-binding molecule and/or the third antigen-binding    molecule is an antibody Fc region having decreased Fcγ    receptor-binding activity;-   [124] the method of any one of [120] to [123], wherein the second    antigen-binding molecule and/or the third antigen-binding molecule    is a bispecific antibody;-   [125] [126] the method of any one of [120] to [124], wherein the    cancer-specific antigen-binding domain of the second antigen-binding    molecule and/or the third antigen-binding molecule is a GPC3-binding    domain;-   [126] the method of any one of [120] and [122] to [125], wherein the    TNF superfamily-binding domain or the TNF receptor    superfamily-binding domain of the second antigen-binding molecule is    a CD137-binding domain or a CD40-binding domain;-   [127] the method of any one of [123] to [126], wherein the T cell    receptor complex-binding domain of the third antigen-binding    molecule is a T cell receptor-binding domain or a CD3-binding    domain; and-   [128] the method of any one of [109] to [127], wherein the cancer    cell is a cell of any cancer selected from the group consisting of    ovarian cancer, gastric cancer, esophageal cancer, pancreatic    cancer, renal cell carcinoma, hepatocellular carcinoma, breast    cancer, malignant melanoma, non-small-cell lung cancer, cervical    cancer, glioblastoma, prostate cancer, neuroblastoma, chronic    lymphocytic leukemia, papillary thyroid cancer, colorectal cancer,    and B-cell non-Hodgkin's lymphoma.

In several embodiments, the present invention relates to methods fortreating or preventing cancer in an individual, the methods comprisingadministering to an individual effective amounts of a T cell-redirectingantigen-binding molecule and an anticancer agent.

Furthermore, the present invention relates to T cell-redirectingantigen-binding molecules, anticancer agents, or pharmaceuticalcompositions comprising a combination of a T cell-redirectingantigen-binding molecule and an anticancer agent, each of which is foruse in combination therapy of the present invention.

Furthermore, the present invention relates to kits for use incombination therapy of the present invention, the kits comprising a Tcell-redirecting antigen-binding molecule and an anticancer agent of thepresent invention.

Furthermore, the present invention relates to use of a Tcell-redirecting antigen-binding molecule and/or an anticancer agent forthe production of pharmaceutical compositions for treating or preventingcancer, the compositions comprising as active ingredients a Tcell-redirecting antigen-binding molecule and/or an anticancer agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a schematic diagram showing cytotoxic activity againstcancer cells by bispecific antibodies that target a cancer antigenexpressed on cancer cells and CD3 expressed on T cells.

FIG. 2-1 presents a schematic diagram showing crosslinking between Tcells caused by crosslinking between CD3s on different T cells via ananti-CD3 antibody.

FIG. 2-2 presents a schematic diagram showing crosslinking between CD3son the same T cell via an anti-CD3 antibody.

FIG. 3 presents a schematic diagram showing that an anti-CTLA4/CD3bispecific antibody causes crosslinking between CTLA4 and CD3 on thesame regulatory T cell, and therefore, does not cause intercellularcrosslinking between an effector T cell and a regulatory T cell. Thisschematic diagram shows a regulatory T cell as the target cell and CTLA4as the antigen, but the target cell and antigen are not limited thereto.

FIG. 4 presents a schematic diagram showing intercellular crosslinkingcaused by crosslinking between CD3 on an effector T cell and CTLA4 on aregulatory T cell via an anti-CTLA4/CD3 bispecific antibody. Thisschematic diagram shows a regulatory T cell as the target cell and CTLA4as the antigen, but the target cell and antigen are not limited thereto.

FIG. 5 presents a graph showing the ADCC activity of the anti-mouseCTLA4 antibody hUH02hUL01-mFa55 on mouse CTLA4-expressing cells.

FIG. 6 presents a graph showing the cytotoxic activity of the anti-mouseCTLA4/anti-mouse CD3 bispecific antibody (hUH02UL01/2C11-F760) on mouseCTLA4-expressing cells.

FIG. 7-1 presents a schematic diagram showing crosslinking between amouse CTLA4-bound bead and a mouse CD3-bound bead by an anti-mouseCTLA4/anti-mouse CD3 bispecific antibody.

FIG. 7-2 presents a graph showing the experiment results of crosslinkingbetween mouse CTLA4-bound beads and mouse CD3-bound beads via anti-mouseCTLA4/anti-mouse CD3 bispecific antibodies (N=3). The graph shows themean values and standard deviations for each of the measurement results.

FIG. 7-3 presents a graph showing the experiment results of crosslinkingbetween mouse CTLA4/mouse CD3-bound beads and mouse CD3-bound beads byanti-mouse CTLA4/anti-mouse CD3 bispecific antibodies. The graph showsthe mean values and standard deviations for each of the measurementresults.

FIG. 7-4 presents a schematic diagram showing crosslinking between amouse CTLA4/mouse CD3-bound bead and a mouse CD3-bound bead by ananti-mouse CTLA4/anti-mouse CD3 bispecific antibody.

FIG. 8 presents a graph showing the in vivo antitumor effects obtainedby intratumoral (i.t.) administration of an anti-mouse CTLA4 antibody(hUH02hUL01-mFa55, ADCC-enhanced antibody) and an anti-mouseCTLA4/anti-mouse CD3 bispecific antibody (hUH02UL01/2C11-F760, a Tcell-redirecting antibody) on mouse colorectal cancer cell line CT26.WT(n=2 for each group; the plot shows the tumor volume of eachindividual).

FIG. 9 presents a graph showing the in vivo antitumor effects obtainedby intratumoral (i.t.) administration and intravenous (i.v.)administration of an anti-mouse CTLA4/anti-mouse CD3 bispecific antibody(hUH02UL01/2C11-F760, a T cell-redirecting antibody) on mouse colorectalcancer cell line CT26.WT (n=5 for each group; the plot shows the meantumor volume+standard deviation of each group).

FIG. 10-1 presents graphs showing the results of analyzing CD4-positivecells based on the expression of CD25 and CD45RA, after a seven-dayreaction of PBMC derived from a healthy person with a control antibody(control) and an anti-human CTLA4/anti-human CD3 bispecific antibody(TRAB).

FIG. 10-2 presents graphs showing the proportion of regulatory T cells(Treg) in CD4-positive T cells, calculated based on the expression ofCD25 and CD45RA, after a seven-day reaction of PBMC derived from ahealthy person with a control antibody (control) and an anti-humanCTLA4/anti-human CD3 bispecific antibody (TRAB).

FIG. 10-3 presents graphs showing the ratio of effector T cells (Teff)to regulatory T cells (Treg) (Teff/Treg) calculated based on theexpression of CD25 and CD45RA, after a seven-day reaction of PBMCderived from a healthy person with a control antibody (control) and ananti-human CTLA4/anti-human CD3 bispecific antibody (TRAB).

FIG. 11-1 presents graphs showing the results of analyzing CD4-positivecells based on the expression of FoxP3 and CD45RA, after a seven-dayreaction of PBMC derived from a healthy person with a control antibody(control) and an anti-human CTLA4/anti-human CD3 bispecific antibody(TRAB).

FIG. 11-2 presents graphs showing the proportion of regulatory T cells(Treg) in CD4-positive T cells, calculated based on the expression ofFoxP3 and CD45RA, after a seven-day reaction of PBMC derived from ahealthy person with a control antibody (control) and an anti-humanCTLA4/anti-human CD3 bispecific antibody (TRAB).

FIG. 11-3 presents graphs showing the ratio of effector T cells (Teff)to regulatory T cells (Treg) (Teff/Treg) calculated based on theexpression of FoxP3 and CD45RA, after a seven-day reaction of PBMCderived from a healthy person with a control antibody (control) and ananti-human CTLA4/anti-human CD3 bispecific antibody (TRAB).

FIG. 12 presents a diagram showing the relationship between the aminoacid residues constituting the Fc regions of IgG, IgG2, IgG3, and IgG4,and Kabat's EU numbering (herein, it is also called the EU INDEX).

FIG. 13 presents graphs showing the results of analyzing CD4-positivecells based on the expression of CD25 and CD45RA, after a four- orsix-day reaction of PBMC derived from a healthy person with ananti-human LAG3/anti-human CD3 bispecific antibody (TRAB).

FIG. 14 presents graphs showing the proportion of regulatory T cells(Treg) in CD4-positive T cells, calculated based on the expression ofCD25 and CD45RA, after a four- or six-day reaction of PBMC derived froma healthy person with an anti-human LAG3/anti-human CD3 bispecificantibody (TRAB).

FIG. 15 presents graphs showing the ratio of effector T cells (Teff) toregulatory T cells (Treg) (Teff/Treg) in CD4-positive T cells,calculated based on the expression of CD25 and CD45RA, after a four- orsix-day reaction of PBMC derived from a healthy person with ananti-human LAG3/anti-human CD3 bispecific antibody (TRAB).

FIG. 16 presents graphs showing the results of analyzing CD4-positivecells based on the expression of CD25 and CD45RA, after a seven-dayreaction of PBMC derived from a healthy person with an anti-humanOX40/anti-human CD3 bispecific antibody (TRAB).

FIG. 17 presents graphs showing the proportion of regulatory T cells(Treg) in CD4-positive T cells, calculated based on the expression ofCD25 and CD45RA, after a seven day reaction of PBMC derived from ahealthy person with an anti-human OX40/anti-human CD3 bispecificantibody (TRAB).

FIG. 18 presents graphs showing the ratio of effector T cells (Teff) toregulatory T cells (Treg) (TeffTreg) in CD4-positive T cells, calculatedbased on the expression of CD25 and CD45RA, after a seven day reactionof PBMC derived from a healthy person with an anti-human OX40/anti-humanCD3 bispecific antibody (TRAB).

FIG. 19 presents a graph showing the results of verifying the effects ofcombined use of the anti-mouse CTLA4/anti-mouse CD3 antibody andgemcitabine in CT26.WT cell-transplanted models. It shows the changes intumor volumes in CT26.WT cell line-transplanted mouse models to whichthe anti-mouse CTLA4/anti-mouse CD3 antibody and gemcitabine have beenadministered as a single agent or in combination. Each point shows themean value of tumor volumes for n=5 per group. Parametric Tukey multiplecomparison tests of the final measurement points indicated in the graphrevealed that significant differences (p<0.05) were observed between thePBS-administered group vs. the mCTLA4/mCD3 antibody-administered group,the PBS-administered group vs. the gemcitabine-administered group, thePBS-administered group vs. the mCTLA4/mCD3antibody+gemcitabine-administered group, and thegemcitabine-administered group vs. the mCTLA4/mCD3antibody+gemcitabine-administered group.

FIG. 20 presents a graph showing the results of verifying the effects ofcombined use of the anti-mouse CTLA4/anti-mouse CD3 antibody andcisplatin in LL/2 (LLC1) cell-transplanted models. It shows the changesin tumor volumes in LL/2 (LLC1) cell line-transplanted mouse models towhich the anti-mouse CTLA4/anti-mouse CD3 antibody and cisplatin havebeen administered as a single agent or in combination. Each point showsthe mean value of tumor volumes for n=5 per group. Parametric Tukeymultiple comparison tests of the final measurement points indicated inthe graph revealed that, while a significant difference (p<0.05) wasobserved between the PBS-administered group vs. the mCTLA4/mCD3antibody+cisplatin-administered group, no significant difference wasobserved for each of the single agent-administered groups with respectto the PBS-administered group.

FIG. 21 presents a graph showing the results of verifying the effects ofcombined use of the anti-mouse CTLA4/anti-mouse CD3 antibody and theanti-mouse PD1 antibody in LL/2 (LLC1) cell-transplanted models. Itshows the changes in tumor volumes in LL/2 (LLC1) cell line-transplantedmouse models to which the anti-mouse CTLA4/anti-mouse CD3 antibody andthe anti-mouse PD1 antibody have been administered as a single agent orin combination. Each point shows the mean value of tumor volumes for n=5per group. Parametric Tukey multiple comparison tests of the finalmeasurement points indicated in the graph revealed that, while asignificant difference (p<0.05) was observed between thePBS-administered group vs. the mCTLA4/mCD3 antibody+mPD1F2-mFa31antibody-administered group, no significant difference was observed foreach of the single agent-administered groups with respect to thePBS-administered group.

FIG. 22 presents a graph showing the results of verifying the effects ofcombined use of the anti-mouse CTLA4/anti-mouse CD3 antibody and theanti-human GPC3/anti-mouse CD3 antibody in LLC1/hGPC3 cell-transplantedmodels. It shows the changes in tumor volumes in LLC1/hGPC3 cellline-transplanted mouse models to which the anti-mouse CTLA4/anti-mouseCD3 antibody and the anti-human GPC3/anti-mouse CD3 antibody have beenadministered as a single agent or in combination. Each point shows themean value of tumor volumes for n=5 per group. Parametric Tukey multiplecomparison tests of the final measurement points indicated in the graphrevealed that, while a significant difference (p<0.05) was observedbetween the PBS-administered group vs. the mCTLA4/mCD3antibody+hGPC3/mCD3 antibody-administered group, no significantdifference was observed for each of the single agent-administered groupswith respect to the PBS-administered group.

FIG. 23 presents a graph showing the results of verifying the effects ofcombined use of the anti-mouse CTLA4/anti-mouse CD3 antibody and theanti-mouse CD137/anti-human GPC3 antibody in CT26/hGPC3cell-transplanted models. It shows the changes in tumor volumes inCT26/hGPC3 cell line-transplanted mouse models to which the anti-mouseCTLA4/anti-mouse CD3 antibody and the anti-mouse CD137/anti-human GPC3antibody have been administered as a single agent or in combination.Each point shows the mean value of tumor volumes for n=5 per group.

MODE FOR CARRYING OUT THE INVENTION Definitions

Herein, unless otherwise defined, the chemical terms and technical termsused in relation to the present invention are regarded as having themeaning generally understood by those skilled in the art. The followingdefinitions are provided to facilitate understanding of the inventiondescribed herein.

Indefinite Article

In the present invention, the indefinite article “a” or “an” refers toone, or two or more (i.e., at least one) grammatical object referred toby the indefinite article. For example, “a component” refers to onecomponent or two or more components.

Amino Acids

Herein, amino acids are expressed in one- or three-letter codes or both:for example, Ala/A, Leu/L, Arg/R, Lys/K, Asn/N, Met/M, Asp/D, Phe/F,Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T, Gly/G, Trp/W, His/H, Tyr/Y,Ile/I, or Val/V.

And/or

As used herein, the term “and/or” includes all combinations formed byappropriately combining “and” and “or”. More specifically, for example,the following variations are included for the phrase “A, B, and/or C”:(a) A, (b) B, (c) C, (d) A and B, (e) A and C, (f) B and C, and (g) Aand B and C. That is, “A, B, and/or C” can be rephrased as “at least oneof A, B, and C”.

EU Numbering and Kabat Numbering

According to the method used in the present invention, the amino acidpositions assigned to antibody CDRs and FRs are specified according toKabat (Sequences of Proteins of Immunological Interest (NationalInstitute of Health, Bethesda, Md., 1987 and 1991)). Herein, when anantigen-binding molecule is an antibody or an antigen-binding fragment,

variable region amino acids are indicated according to Kabat numbering,while constant region amino acids are indicated according to EUnumbering based on Kabat's amino acid positions.

Cancer

Herein, the terms “cancer”, “carcinoma”, “tumor”, “neoplasm”, and suchare not differentiated from each other and are mutually interchangeable.

Antigen-Binding Molecules

In several embodiments, “antigen-binding molecules” according to thepresent invention are not particularly limited as long as they aremolecules that comprise a “binding domain” of the present invention, andthey may further comprise a peptide or protein having a length of aboutfive amino acids or more. The peptide and protein are not limited tothose derived from a living organism, and for example, they may be apolypeptide produced from an artificially designed sequence. They mayalso be any of a naturally-occurring polypeptide, synthetic polypeptide,recombinant polypeptide, and such.

A favorable example of an antigen-binding molecule of the presentinvention is an antigen-binding molecule that comprises an FcRn-bindingdomain contained in an antibody Fc region. As a method for extending theblood half-life of a protein administered to a living body, the methodof adding an FcRn-binding domain of an antibody to the protein ofinterest and utilizing the function of FcRn-mediated recycling is wellknown.

FcRn-Binding Domains

In several embodiments, the “FcRn-binding domain” according to thepresent invention is not particularly limited as long as it has bindingactivity to FcRn, and it may be a domain that directly or indirectlybinds to FcRn. Examples of the domain that directly binds to FcRninclude antibody variable regions whose antigens are FcRn, Fab, antibodyFc regions, fragments thereof, albumin, albumin domain 3, human serumalbumin (HSA), transferrin and such. Furthermore, an example of thedomain that indirectly binds to FcRn includes a domain that has bindingactivity toward the aforementioned domain that directly binds to FcRn. Apreferred embodiment of the present invention includes antibody Fcregions or fragments containing an FcRn-binding region of an Fc region.Herein, for example, an Fc region derived from a naturally-occurring IgGmay be used as the “Fc region”. A naturally-occurring IgG means apolypeptide that comprises the same amino acid sequence as an IgG foundin nature, and belongs to a class of antibodies substantially encoded byimmunoglobulin gamma genes. A naturally-occurring human IgG means, forexample, a naturally-occurring human IgG1, a naturally-occurring humanIgG2, a naturally-occurring human IgG3, or a naturally-occurring humanIgG4. Naturally-occurring IgGs also include mutants and such thatnaturally generate therefrom. A plurality of allotype sequences thatresult from genetic polymorphism have been described in Sequences ofProteins of Immunological Interest, NIH Publication No. 91-3242 for thehuman IgG1, human IgG2, human IgG3, and human IgG4 antibody constantregion, and any of the sequences may be used in the present invention.In particular, the amino acid sequence of positions 356 to 358 accordingto EU numbering may be DEL or EEM for the human IgG1 sequence.

Fc Regions

Existing antibody Fc regions are, for example, IgA1, IgA2, IgD, IgE,IgG1, IgG2, IgG3, IgG4, and IgM-type Fc regions. For example, an Fcregion derived from a naturally-occurring human IgG antibody can be usedas the antibody Fc region of the present invention. Fc regions derivedfrom a constant region of a naturally-occurring IgG, or morespecifically, a constant region derived from a naturally-occurring humanIgG1 (SEQ ID NO: 1), a constant region derived from anaturally-occurring human IgG2 (SEQ ID NO: 2), a constant region derivedfrom a naturally-occurring human IgG3 (SEQ ID NO: 3), and a constantregion derived from a naturally-occurring human IgG4 (SEQ ID NO: 4), canbe used as an Fc region of the present invention. Mutants and such thatnaturally generate therefrom are also included in thenaturally-occurring IgG constant regions.

Such antibody Fc regions can be suitably obtained, for example, bypartial digestion of antibodies such as monoclonal antibodies using aprotease such as pepsin, then adsorption of the resulting fragments ontoa protein A column or a protein G column, and subsequent elution usingan appropriate elution buffer and such. The protease is not particularlylimited as long as it can digest an antibody such as a monoclonalantibody by appropriately establishing the enzyme reaction conditionssuch as pH, and examples include pepsin and ficin.

The isotype of an antibody is determined by the structure of theconstant region. The constant region of isotypes IgG1, IgG2, IgG3, andIgG4 is called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acidsequences of polypeptides constituting the Fc regions of human Cγ1, Cγ2,Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 5, 6, 7, and 8. Therelationship between amino acid residues constituting each of theseamino acid sequences and Kabat's EU numbering (herein, also referred toas EU INDEX) is shown in FIG. 12.

An Fc region refers to a region that excludes F(ab′)2 which contains twolight chains and two heavy chains containing part of the constant regionbetween the CH1 domain and the CH2 domain such that the disulfide bondsbetween the chains are formed between the two heavy chains. Fc regionsforming the antigen-binding molecules disclosed herein can be obtainedsuitably by partially digesting the IgG1, IgG2, IgG3, or IgG4 monoclonalantibodies or the like using a protease such as pepsin, and thenre-eluting fractions adsorbed to the protein A column. The protease isnot particularly limited as long as it can digest a full-length antibodyin a restrictive manner to produce F(ab′)2 by appropriately establishingthe enzyme reaction conditions such as pH. Such proteases include, forexample, pepsin and ficin.

Fcγ Receptors

In several embodiments, a domain with decreased Fcγ receptor-bindingactivity is particularly preferred as the FcRn-binding domain of thepresent invention. Here, an Fcγ receptor (herein, also denoted as Fcγreceptor, FcγR, or FcgR) refers to a receptor that can bind to the Fcregion of IgG1, IgG2, IgG3, or IgG4, and includes all members belongingto the family of proteins substantially encoded by Fcγ receptor genes.In humans, this family includes, but is not limited to, FcγRI (CD64)including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) includingisoforms FcγRIIa (including allotypes H131 (type H) and R131 (type R),FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16) including isoforms FcγRIIIa (including allotypes V158 and F158)and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); aswell as any undiscovered human FcγRs, and FcγR isoforms or allotypes.FcγRs include, but are not limited to, those derived from humans, mice,rats, rabbits, and monkeys, and may be derived from any organism. MouseFcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32),FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscoveredmouse FcγRs, and FcγR isoforms or allotypes. Suitable examples of suchFcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb(CD32), FcγRIIIa (CD16) and/or FcγRIIIb (CD16).

Activating receptors which carry an immunoreceptor tyrosine-basedactivation motif (ITAM) and inhibitory receptors which carry animmunoreceptor tyrosine-based inhibitory motif (ITIM) are present amongFcγRs. FcγRs are categorized into activating FcγRs: FcγRI, FcγRIIa R,FcγRIIa H, FcγRIIIa, and FcγRIIIb, and inhibitory FcγR: FcγRIIb.

The polynucleotide sequence and amino acid sequence of FcγRI are shownin NM_000566.3 and NP_000557.1, respectively; the polynucleotidesequence and amino acid sequence of FcγRIIa are shown in BC020823.1 andAAH20823.1, respectively; the polynucleotide sequence and amino acidsequence of FcγRIIb are shown in BC146678.1 and AAI46679.1,respectively; the polynucleotide sequence and amino acid sequence ofFcγRIIIa are shown in BC033678.1 and AAH33678.1, respectively; and thepolynucleotide sequence and amino acid sequence of FcγRIIIb are shown inBC128562.1 and AAI28563.1, respectively (RefSeq accession number). Thereare two types of gene polymorphisms for FcγRIIa, where the amino acid atposition 131 of FcγRIIa is substituted with histidine (type H) orarginine (type R) (J. Exp. Med, 172, 19-25, 1990). Furthermore, thereare two types of gene polymorphisms for FcγRIIb, where the amino acid atposition 232 of FcγRIIb is substituted with isoleucine (type I) orthreonine (type T) (Arthritis. Rheum. 46: 1242-1254 (2002)). Inaddition, there are two types of gene polymorphisms for FcγRIIIa, wherethe amino acid at position 158 of FcγRIIIa is substituted with valine(type V) or phenylalanine (type F) (J. Clin. Invest. 100(5): 1059-1070(1997)). There are also two types of gene polymorphisms for FcγRIIIb,which are type NA1 and type NA2 (J. Clin. Invest. 85: 1287-1295 (1990)).

Whether the binding activity to an Fcγ receptor is decreased can beconfirmed by well-known methods such as FACS, ELISA format, screening byAmplified Luminescent Proximity Homogeneous Assay (ALPHA), surfaceplasmon resonance (SPR)-based BIACORE method, and others (Proc. Natl.Acad. Sci. USA (2006) 103(11), 4005-4010).

ALPHA screening is performed with ALPHA technology which uses two beads,a donor and an acceptor bead, based on the following principle.Luminescent signals are detected only when molecules bound to donorbeads interact biologically with molecules bound to the acceptor beads,and the two beads are in close proximity to each other. Thelaser-excited photosensitizer within the donor beads converts ambientoxygen to excited-state singlet oxygen. Singlet oxygen is dispersedaround the donor beads; and when it reaches the adjacent acceptor beads,a chemiluminescent reaction is induced within the beads, and light isultimately emitted. When molecules bound to the donor beads do notinteract with molecules bound to the acceptor beads, thechemiluminescent reaction does not take place because singlet oxygenproduced by the donor beads does not reach the acceptor beads.

For example, when an antigen-binding molecule contains an antibody Fcregion as the FcRn-binding domain, an antigen-binding molecule having awild-type Fc region and an antigen-binding molecule having a mutant Fcregion produced by adding amino acid mutations to change the binding toan Fcγ receptor are prepared, a biotinylated antigen-binding molecule isbound to the donor beads, and an Fcγ receptor tagged with glutathione Stransferase (GST) is bound to the acceptor beads. In the presence of anantigen-binding molecule having a mutant Fc region, the antigen-bindingmolecule having a wild-type Fc region interacts with the Fcγ receptorand produces 520-620 nm signals. When the antigen-binding moleculehaving a mutant Fc region is untagged, it competes with theantigen-binding molecule having a wild-type Fc region for interactionwith the Fcγ receptor. The relative binding affinity can be determinedby quantifying the decrease in fluorescence observed as a result of thecompetition. Biotinylation of antigen-binding molecules usingSulfo-NHS-biotin and such is well known. As a method for tagging an Fcγreceptor with GST, the method of expressing the Fcγ receptor and GST ina cell carrying a vector that can express a fusion gene produced byfusing a polynucleotide encoding the Fcγ receptor in frame with aGST-encoding polynucleotide, and purifying it using a glutathione columncan be appropriately adopted. The obtained signals are suitablyanalyzed, for example, by fitting them into a one-site competition modelthat utilizes a non-linear regression analysis with software such asGRAPHPAD PRISM (GraphPad, San Diego).

One of the substances (ligand) observed for interaction is immobilizedonto a gold thin film on a sensor chip, and by shining light from thereverse side of the sensor chip so that total reflection takes place atthe interface between the gold thin film and glass, a portion withreduced reflection intensity is formed in part of the reflected light(SPR signal). The other substance (analyte) observed for interaction ismade to flow over the sensor chip surface; and when the ligand binds tothe analyte, the mass of the immobilized ligand molecule increases andthe refractive index of the solvent on the sensor chip surface changes.The position of the SPR signal shifts as a result of this change in therefractive index (reversely, the signal position returns if this bindingdissociates). The Biacore system shows the amount of shift mentionedabove, or more specifically the time variable of mass, by plotting thechange in mass on the sensor chip surface on the vertical axis as themeasurement data (sensorgram). Kinetic parameters such as associationrate constant (ka) and dissociation rate constant (kd) are determinedfrom the curve in the sensorgram, and the affinity (KD) is determinedfrom the ratio of these constants. In the BIACORE method, a method formeasuring inhibition is also suitably used. An example of the method formeasuring inhibition is described in Proc. Natl. Acad. Sci USA (2006)103 (11): 4005-4010.

Herein, in several embodiments, “decreased Fcγ receptor-bindingactivity” means that, for example, based on the above-describedanalytical method, the binding activity of the test antigen-bindingmolecule is 50% or less, preferably 45% or less, 40% or less, 35% orless, 30% or less, 20% or less, 15% or less, or particularly preferably10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,4% or less, 3% or less, 2% or less, or 1% or less as compared to thebinding activity of the control antigen-binding molecule containing anFc region.

For the control antigen-binding molecule, antigen-binding moleculeshaving, for example, a domain comprising an Fc region of a monoclonalIgG1, IgG2, IgG3, or IgG4 antibody may be suitably used. The structuresof the Fc regions are shown in SEQ ID NO: 1 (A is added to the Nterminus of RefSeq Accession No. AAC82527.1), SEQ ID NO: 2 (A is addedto the N terminus of RefSeq Accession No. AAB59393.1), SEQ ID NO: 3 (Ais added to the N terminus of RefSeq Accession No. CAA27268.1), and SEQID NO: 4 (A is added to the N terminus of RefSeq Accession No.AAB59394.1). Further, when an antigen-binding molecule containing amutant of an Fc region of a particular antibody isotype is used as thetest substance, the effect of a mutation possessed by the mutant on theFcγ receptor-binding activity is tested by using as a control anantigen-binding molecule having an Fc region of an antibody of thatparticular isotype. In this way, antigen-binding molecules containing anFc region mutant whose binding activity toward the Fcγ receptor verifiedto be decreased are suitably produced.

Examples of such mutants include mutants with a 231A-238S deletion (WO2009/011941), or C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34,11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54), C226S,C229S, E233P, L234V, or L235A (Blood (2007) 109, 1185-1192) mutants,where the amino acids are specified by EU numbering.

That is, suitable examples include antigen-binding molecules having anFc region in which any of the amino acids at positions 220, 226, 229,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267,269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331,and 332 specified according to EU numbering has been substituted in theamino acids constituting the Fc region of an antibody of a specificisotype. The isotype of the antibody from which the Fc region originatesis not particularly limited, and the Fc region derived from an IgG1,IgG2, IgG3, or IgG4 monoclonal antibody can be used appropriately, andthe Fc region derived from a naturally-occurring human IgG1 antibody issuitably used.

For example, an antigen-binding molecule having an Fc region thatcomprises any substitution specified below based on EU numbering fromamong amino acids constituting the IgG1 antibody Fc region (wherein thenumber indicates the position of the amino acid residue specifiedaccording to EU numbering, the one-letter amino acid code positionedbefore the number indicates the amino acid residue before thesubstitution, and the one-letter amino acid code positioned after thenumber indicates the amino acid residue after the substitution):

-   -   (a) L234F, L235E, P331S    -   (b) C226S, C229S, P238S    -   (c) C226S, C229S    -   (d) C226S, C229S, E233P, L234V, L235A;        or an Fc region lacking the amino acid sequence of positions 231        to 238 from among amino acids constituting the IgG1 antibody Fc        region may be appropriately used.

Furthermore, antigen-binding molecules having an Fc region thatcomprises any substitution specified below based on EU numbering fromamong amino acids constituting the IgG2 antibody Fc region (wherein thenumber indicates the position of the amino acid residue specifiedaccording to EU numbering, the one-letter amino acid code positionedbefore the number indicates the amino acid residue before thesubstitution, and the one-letter amino acid code positioned after thenumber indicates the amino acid residue after the substitution):

-   -   (e) H268Q, V309L, A330S, P331S    -   (f) V234A    -   (g) G237A    -   (h) V234A, G237A    -   (i) A235E, G237A    -   (j) V234A, A235E, G237A    -   may be appropriately used.

Furthermore, antigen-binding molecules having an Fc region thatcomprises any substitution specified below based on EU numbering fromamong amino acids constituting the IgG3 antibody Fc region (wherein thenumber indicates the position of the amino acid residue specifiedaccording to EU numbering, the one-letter amino acid code positionedbefore the number indicates the amino acid residue before thesubstitution, and the one-letter amino acid code positioned after thenumber indicates the amino acid residue after the substitution):

-   -   (k) F241A    -   (l) D265A    -   (m) V264A    -   may be appropriately used.

Furthermore, antigen-binding molecules having an Fc region thatcomprises any substitution specified below based on EU numbering fromamong amino acids constituting the IgG4 antibody Fc region (wherein thenumber indicates the position of the amino acid residue specifiedaccording to EU numbering, the one-letter amino acid code positionedbefore the number indicates the amino acid residue before thesubstitution, and the one-letter amino acid code positioned after thenumber indicates the amino acid residue after the substitution):

-   -   (n) L235A, G237A, E318A    -   (o) L235E    -   (p) F234A, L235A    -   may be appropriately used.

Other preferred examples include antigen-binding molecules having an Fcregion in which any of the amino acids at positions 233, 234, 235, 236,237, 327, 330, and 331 specified according to EU numbering in the aminoacids constituting the Fc region of a naturally-occurring human IgG1antibody is substituted with amino acids of corresponding EU numberingin the corresponding IgG2 or IgG4.

Other preferred examples suitably include antigen-binding moleculeshaving an Fc region in which any one or more of the amino acids atpositions 234, 235, and 297 specified according to EU numbering in theamino acids constituting the Fc region of a naturally-occurring humanIgG1 antibody are substituted by other amino acids. The type of aminoacid present after substitution is not particularly limited, and anantigen-binding molecule having an Fc region in which any one or more ofthe amino acids at positions 234, 235, and 297 are substituted withalanine is particularly preferred.

Other preferred examples suitably include antigen-binding moleculeshaving an Fc region in which the amino acid at position 265 specifiedaccording to EU numbering in the amino acids constituting an IgG1antibody Fc region is substituted by another amino acid. The type ofamino acid present after substitution is not particularly limited, andan antigen-binding molecule having an Fc region in which the amino acidat position 265 is substituted with alanine is particularly preferred.

Antigen-Binding Domains

“A domain that binds to a molecule expressed on the surface of a cellhaving immune response-suppressing function”, “a T cell receptorcomplex-binding domain”, “a cancer-specific antigen-binding domain”, “atumor necrosis factor (TNF) superfamily-binding domain”, and “a tumornecrosis factor (TNF) receptor superfamily-binding domain” (hereinafter,these binding domains are collectively referred to as “antigen-bindingdomains”) which are included in antigen-binding molecules of the presentinvention mean regions that specifically bind to all or a portion oftheir respective antigens. An example of such binding domain is a regioncomprising the antigen-binding region of an antibody. When the molecularweight of the antigen is large, the antigen-binding region of theantibody can bind only to a specific portion of the antigen. Thisspecific portion is called an epitope. One or more of antibody variabledomains may provide an antigen-binding domain. Preferably,antigen-binding domains comprise an antibody light chain variable region(VL) and an antibody heavy chain variable region (VH). Suitable examplesof such antigen-binding domains include “single chain Fv (scFv)”,“single chain antibody”, “Fv”, “single chain Fv2 (scFv2)”, “Fab”,“F(ab′)2”, or such.

In several embodiments, “a cell having immune response-suppressingfunction” is not particularly limited as long as it has a function ofsuppressing an immune response, and examples include regulatory T cells(Treg), exhausted T cells, myeloma-derived stromal cells (MDSC),tumor-associated macrophages (TAM), induced regulatory T cells (Trl),tumor-associated dendritic cells (TADC), tumor-associated neutrophils(TAN), cancer-associated fibroblasts (CAF), regulatory B cells (Breg),and such. In particular, regulatory T cells and exhausted T cells arepreferable as the cells of interest. Specific examples of the moleculesexpressed on the surface of such cells having immuneresponse-suppressing functions include CTLA4, PD1, TIM3, LAG-3, CD244(2B4), CD160, GARP, OX40, CD137 (4-1BB), CD25, VISTA, VISATA, BTLA,TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6, CD39, CD73, CD4, CD18, CD49b,CD1d, CD5, CD21, TIM1, CD19, CD20, CD23, CD24, CD38, CD93, IgM,B220(CD45R), CD317, PD-L1, CD11b, Ly6G, ICAM-1, FAP, PDGFR, Podoplanin,TIGIT, and such. Among these molecules, examples of favorable moleculesfor binding targets of the binding domains of the present inventioninclude CTLA4, TIM3, LAG3, CD137 (4-1BB), CD25, CCR5, CCR6, CD38, andTIGIT, which are cell surface molecules specifically expressed in cellfractions (CD4+, CD25^(high), and CD45RA−) that have been reported tohave high immune response-suppressing functions. Examples of favorablemolecules for binding targets of the binding domains of the presentinvention include CTLA4, LAG3, and OX40 in particular.

In several embodiments, “regulatory T cell” according to the presentinvention means a type of T cell in charge of inhibitory regulation ofan immune response. This cell plays an important role in the negativeregulation mechanism for suppressing excessive immune responses and inhomeostasis of immunity, and is classified into two types of regulatoryT cells (Tregs) which express CD4 or CD8. CD4 Tregs are classified intoendogenous Treg cells (natural Tregs or nTregs) which constitutivelyexpress CD25 and FoxP3, and adaptive or inducible Tregs (iTregs) whichhave low self-recognition ability and which are differentiated fromnaive CD4-positive T cells. Existing iTregs include Foxp3+ Treg andFop3− Treg, and Fop3− Treg is called a Type I Treg (Trl). CD4+CD25+LAG3+Treg has been identified as a Treg having properties very similar to Trl(Proc Natl Acad Sci USA.2009). Furthermore, CD127 expression is known tobe decreased in Treg cells, and the fraction containingCD127loCD25hi-int (population showing low CD127 expression and high tointermediate CD25 expression) includes all of the Foxp3-positive Tregpopulation. CD8 Tregs can also be separated into endogenous Tregs andinducible Tregs. The former are classified as CD8+CD122+ Treg, and thelatter are classified as Qa-1a-restricted CD8+ Treg. Tregs are known toexpress regulatory molecules, and have increased expression levels ofCTLA4, PD1, TIM3, LAG3, and the like, and such molecules are favorableas the molecules to which the binding domains of the antigen-bindingmolecules of the present invention bind.

Furthermore, in several embodiments, “exhausted T cell” according to thepresent invention means a T cell whose cytokine production function andeffector function have been markedly weakened by continuous stimulationby antigens, and proliferation ability and long-term survival abilityhave become low. These exhausted T cells produce regulatory receptorssuch as PD1 and regulatory cytokines; therefore, they not only becomedysfunctional, but also act suppressive towards immune responses. Inexhausted T cells, expression of PD1 is mainly increased (Nature 439,682-687 (2006)). In addition to PD1, expression of molecules such asLAG-3, CD244 (2B4), CD160, TIM-3, and CTLA-4 are also increased (NatureImmunology Volume: 12, Pages: 492-499 Year published: (2011)). Thesemolecules are favorable as the molecules to which the binding domains ofthe antigen-binding molecules of the present invention bind.

A “T cell-receptor complex” may be a T cell receptor itself, or anadaptor molecule constituting a T cell-receptor complex together with aT cell receptor. CD3 is suitable as an adaptor molecule.

For the T cell receptor, an epitope to which the T cell receptor bindingdomain binds may be a variable region or a constant region, but anepitope present in the constant region is preferred. Examples of theconstant region sequence include the T cell receptor α chain of RefSeqAccession No. CAA26636.1 (SEQ ID NO: 9), the T cell receptor β chain ofRefSeq Accession No. C25777 (SEQ ID NO: 10), the T cell receptor γ1chain of RefSeq Accession No. A26659 (SEQ ID NO: 11), the T cellreceptor γ2 chain of RefSeq Accession No. AAB63312.1 (SEQ ID NO: 12),and the T cell receptor^(δ) chain of RefSeq Accession No. AAA61033.1(SEQ ID NO: 13).

In several embodiments, when the “CD3-binding domain” is used as the Tcell receptor complex-binding domain in the present invention, theCD3-binding domain may be provided by one or more antibody variabledomains. Preferably, the CD3-binding domain includes a light chainvariable region (VL) and a heavy chain variable region (VH) of a CD3antibody. Suitable examples of such CD3-binding domains include “singlechain Fv (scFv)”, “single chain antibody”, “Fv”, “single chain Fv 2(scFv2)”, “Fab”, “F(ab′)2”, and such.

In several embodiments, the CD3-binding domain of the present inventionmay be those that bind to any epitope as long as the epitope exists inthe γ-chain, ^(δ)-chain, or ε-chain sequence constituting human CD3. Inthe present invention, preferably, a CD3-binding domain that comprises alight chain variable region (VL) of a CD3 antibody and a heavy chainvariable region (VH) of a CD3 antibody, and which binds to an epitopepresent in the extracellular region of the c chain of the human CD3complex, is suitably used. For such CD3-binding domain, a CD3-bindingdomain comprising the light chain variable region (VL) and heavy chainvariable region (VH) of the OKT3 antibody (Proc. Natl. Acad. Sci. USA(1980) 77, 4914-4917) or various known CD3 antibodies is suitably used.A CD3-binding domain derived from a CD3 antibody that has the desiredproperties and is obtained by immunizing a desired animal with theγ-chain, ^(δ)-chain, or ε-chain constituting the human CD3 by theabove-mentioned method may be appropriately used. Human antibodies andappropriately humanized antibodies as described below may be suitablyused as the CD3 antibody that serves as the origin for the CD3-bindingdomain. For the structure of the CD3-constituting γ-chain, ^(δ)-chain,or ε-chain, their polynucleotide sequences are shown in SEQ ID NOs: 14(NM_000073.2), 16 (NM_000732.4), and 18 (NM_000733.3), and theirpolypeptide sequences are shown in SEQ ID NOs: 15 (NP_000064.1), 17(NP_000723.1), and 19 (NP_000724.1) (the RefSeq accession number isshown in parentheses).

In several embodiments, a “cancer-specific antigen” refers to an antigenexpressed by cancer cells, which enables discrimination between cancercells and healthy cells; and for example, it includes antigens that areexpressed as cells become malignant, or abnormal sugar chains thatappear on protein molecules or cell surface when cells become cancerous.Specific examples include GPC3 which belongs to the GPI anchor-typereceptor family and is expressed in several cancers including livercancer (Int. J. Cancer. (2003) 103 (4): 455-65); ALK receptor(pleiotrophin receptor); pleiotrophin; KS 1/4 pancreatic cancer antigen;ovarian cancer antigen (CA125); prostatic acid phosphate;prostate-specific antigen (PSA); melanoma-associated antigen p97;melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA);prostate-specific membrane antigen; carcinoembryonic antigen (CEA);polymorphic epithelial mucin antigen; human milk fat globule antigen;colorectal tumor-associated antigens such as CEA, TAG-72, C017-1A, GICA19-9, CTA-1, and LEA; Burkitt's lymphoma antigen-38.13; CD19; humanB-lymphoma antigen-CD20; CD33; melanoma-specific antigens such asganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3;tumor-specific transplantation type cell-surface antigen (TSTA);virus-induced tumor antigens including T antigen and envelope antigensof DNA tumor viruses and RNA tumor viruses; CEA of colon; oncofetalantigens such as 5T4 oncofetal trophoblast glycoprotein and bladdertumor oncofetal antigen; α-fetoprotein; differentiation antigens such ashuman lung cancer antigens L6 and L20; antigens of fibrosarcoma; humanleukemia T cell antigen-Gp37; breast cancer antigens such asneoglycoprotein, sphingolipids, and EGFR (epidermal growth factorreceptor); NY-BR-16; NY-BR-16 and HER2 antigen (p185HER2); polymorphicepithelial mucin (PEM); malignant human lymphocyte antigen-APO-1;differentiation antigens such as I antigen found in fetal erythrocytes;primary endoderm I antigen found in adult erythrocytes; I(Ma) found inpreimplantation embryos and gastric cancer; M18 and M39 found in mammaryepithelium; SSEA-1, VEP8, VEP9, Myl, and VIM-D5 found in myeloid cells;D156-22 found in colorectal cancer; TRA-1-85 (blood group H); SCP-1found in testis and ovarian cancer; C14 found in rectal cancer; F3 foundin lung cancer; AH6 found in gastric cancer; Y hapten; Ley found inembryonal carcinoma cells; TL5 (blood group A); EGF receptor found inA431 cells; E1 series (blood group B) found in pancreatic cancer; FC10.2 found in embryonal carcinoma cells; gastric cancer antigen; CO-514(blood group Lea) found in adenocarcinomas; NS-10 found inadenocarcinomas; CO-43 (blood group Leb); G49 found in EGF receptor ofA431 cells; MH2 (blood group ALeb/Ley) found in colon cancer; 19.9 foundin colon cancer; gastric cancer mucins; T5A7 found in myeloid cells; R24found in melanoma; 4.2, GD3, DI.1, OFA-1, GM2, OFA-2, GD2, andM1:22:25:8 found in embryonal carcinoma cells; SSEA-3 and SSEA-4 foundin 4 to 8-cell stage embryos; subcutaneous T cell lymphoma antigen;MART-1 antigen; sialyl Tn (STn) antigen; colon cancer antigen NY-CO-45;lung cancer antigen NY-LU-12 variant A; adenocarcinoma antigen ARTI;paraneoplastic associated brain-testis-cancer antigen (onconeuronalantigen MA2; paraneoplastic neuronal antigen); neuro-oncological ventralantigen 2 (NOVA2); hemocyte carcinoma antigen gene 520; tumor-associatedantigen CO-029; tumor-associated antigens MAGE-C1 (cancer/testis antigenCT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a,MAGE-4b and MAGE-X2; Cancer-Testis Antigen (NY-EOS-1); YKL-40, fragmentsof any of the aforementioned polypeptides, or structures produced bymodification thereof (for example, the above-mentioned modifiedphosphate group or sugar chain); EpCAM; EREG; CA19-9; CA15-3; sialylSSEA-1(SLX); HER2; PSMA; CEA; and CLEC12A. Cancer-specific antigenswhich are binding targets of the cancer-specific antigen-binding domainsof the present invention are, in particular, preferably those expressedon cell surface, and examples of such cancer-specific antigens includeGPC3, CD 19, CD20, EGFR, HER2, EpCAM, and EREG.

In several embodiments, as factors belonging to the “TNF superfamily” orthe “TNF receptor superfamily”, ligands having a trimeric structure andreceptors with a trimeric structure to which the ligands bind, whichcontribute to activation of various immune cells, are known (Nat. Rev.Immunol., 2012, 12, 339-51). Examples of factors belonging to the TNFsuperfamily or the TNF receptor superfamily include CD137, CD137L, CD40,CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153,GITR, and GITRL. Preferred factors include, for example, CD137 and CD40.A more preferred factor is, for example, CD137.

Antibdies

In several embodiments, an “antigen-binding molecule” of the presentinvention includes an antibody. Furthermore, a part of a preferredembodiment of the present invention includes an antibody comprising anantibody variable region described herein.

Herein, in several embodiments, an “antibody” refers to a naturallyoccurring immunoglobulin or an immunoglobulin produced by partial orcomplete synthesis. Antibodies can be isolated from natural sources suchas naturally-occurring plasma and serum, or culture supernatants ofantibody-producing hybridoma cells. Alternatively, antibodies can bepartially or completely synthesized using techniques such as geneticrecombination. Suitable examples of the antibodies include antibodies ofan immunoglobulin isotype or subclass of such isotype. Known humanimmunoglobulins include those of the following nine classes (isotypes):IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of theseisotypes, antibodies of the present invention include IgG1, IgG2, IgG3,and IgG4.

Methods for producing antibodies having the desired binding activity areknown to those skilled in the art, and the antibodies may be obtained aspolyclonal or monoclonal antibodies. Monoclonal antibodies derived frommammals may be suitably produced as the antibodies of the presentinvention. Such mammalian-derived monoclonal antibodies includeantibodies produced by hybridomas and antibodies produced by host cellstransformed with an expression vector carrying an antibody gene bygenetic engineering techniques.

There is no particular limitation on the mammals to be immunized forobtaining antibodies. It is preferable to select the mammals byconsidering its compatibility with the parent cells to be used in cellfusion for hybridoma production. In general, rabbits, monkeys, androdents such as mice, rats, and hamsters are suitably used.

The above animals are immunized with a sensitizing antigen by knownmethods. Generally performed immunization methods include, for example,intraperitoneal or subcutaneous injection of a sensitizing antigen intomammals. Specifically, a sensitizing antigen is appropriately dilutedwith Phosphate-Buffered Saline (PBS), physiological saline, or the like.If desired, a conventional adjuvant such as Freund's complete adjuvantis mixed with the antigen, and the mixture is emulsified. Then, thesensitizing antigen is administered to mammals several times at 4- to21-day intervals. Appropriate carriers may be used in immunization withthe sensitizing antigen. In particular, when a low-molecular-weightpartial peptide is used as the sensitizing antigen, it is sometimesdesirable to couple the sensitizing antigen peptide to a carrier proteinsuch as albumin or keyhole limpet hemocyanin for immunization.

Alternatively, hybridomas producing a desired antibody can be preparedusing DNA immunization as mentioned below. DNA immunization is animmunization method that confers immunostimulation by expressing asensitizing antigen in an animal immunized as a result of administeringa vector DNA constructed to allow expression of an antigenprotein-encoding gene in the animal. As compared to conventionalimmunization methods in which a protein antigen is administered toanimals to be immunized, DNA immunization is expected to be superior inthat:

-   -   immunostimulation can be provided while retaining the structure        of a membrane protein; and    -   there is no need to purify the antigen for immunization.

In order to prepare a monoclonal antibody of the present invention usingDNA immunization, first, a DNA expressing an antigen protein isadministered to animals to be immunized. The antigen protein-encodingDNA can be synthesized by known methods such as PCR. The obtained DNA isinserted into an appropriate expression vector, and then this isadministered to animals to be immunized. Preferably used expressionvectors include, for example, commercially-available expression vectorssuch as pcDNA3.1. Vectors can be administered to an organism usingconventional methods. For example, DNA immunization is performed byusing a gene gun to introduce expression vector-coated gold particlesinto cells in the body of animals to be immunized.

After immunizing mammals as described above, an increase in the titer ofan antigen-binding antibody is confirmed in the serum. Then, immunecells are collected from the mammals, and then subjected to cell fusion.In particular, splenocytes are preferably used as immune cells.

A mammalian myeloma cell is used as a cell to be fused with theabove-mentioned immune cells. The myeloma cells preferably comprise asuitable selection marker for screening. A selection marker conferscharacteristics to cells for their survival (or death) under a specificculture condition. Hypoxanthine-guanine phosphoribosyltransferasedeficiency (hereinafter abbreviated as HGPRT deficiency) and thymidinekinase deficiency (hereinafter abbreviated as TK deficiency) are knownas selection markers. Cells with HGPRT or TK deficiency havehypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviatedas HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in a HATselection medium, and are thus killed. However, when the cells are fusedwith normal cells, they can continue DNA synthesis using the salvagepathway of the normal cells, and therefore they can grow even in the HATselection medium.

HGPRT-deficient and TK-deficient cells can be selected in a mediumcontaining 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG),or 5′-bromodeoxyuridine. Normal cells are killed because theyincorporate these pyrimidine analogs into their DNA. Meanwhile, cellsthat are deficient in these enzymes can survive in the selection medium,since they cannot incorporate these pyrimidine analogs. In addition, aselection marker referred to as G418 resistance provided by theneomycin-resistant gene confers resistance to 2-deoxystreptamineantibiotics (gentamycin analogs). Various types of myeloma cells thatare suitable for cell fusion are known.

For example, myeloma cells including the following cells can bepreferably used:

-   -   P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550);    -   P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology        (1978)81, 1-7);    -   NS-1 (C. Eur. J. Immunol. (1976)6 (7), 511-519);    -   MPC-11 (Cell (1976) 8 (3), 405-415);    -   SP2/0 (Nature (1978) 276 (5685), 269-270);    -   FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);    -   S194/5.XXO.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);    -   R210 (Nature (1979) 277 (5692), 131-133), etc.

Cell fusions between the immunocytes and myeloma cells are essentiallycarried out using known methods, for example, a method by Kohler andMilstein et al. (Methods Enzymol. (1981) 73: 3-46).

More specifically, cell fusion can be carried out, for example, in aconventional culture medium in the presence of a cell fusion-promotingagent. The fusion-promoting agents include, for example, polyethyleneglycol (PEG) and Sendai virus (HVJ). If required, an auxiliary substancesuch as dimethyl sulfoxide is also added to improve fusion efficiency.

The ratio of immunocytes to myeloma cells may be arbitrarily set,preferably, for example, one myeloma cell for every one to tenimmunocytes. Culture media to be used for cell fusions include, forexample, media that are suitable for the growth of myeloma cell lines,such as RPMI1640 medium and MEM medium, and other conventional culturemedium used for this type of cell culture. In addition, serumsupplements such as fetal calf serum (FCS) may be preferably added tothe culture medium.

For cell fusion, predetermined amounts of the above immune cells andmyeloma cells are mixed well in the above culture medium. Then, a PEGsolution (for example, the average molecular weight is about 1,000 to6,000) prewarmed to about 37° C. is added thereto at a concentration ofgenerally 30% to 60% (w/v). The mixed solution is gently mixed toproduce desired fusion cells (hybridomas). Then, an appropriate culturemedium mentioned above is gradually added to the cells, and this isrepeatedly centrifuged to remove the supernatant. Thus, cell fusionagents and such which are unfavorable to hybridoma growth can beremoved.

The hybridomas thus obtained can be selected by culture using aconventional selective medium, for example, HAT medium (a culture mediumcontaining hypoxanthine, aminopterin, and thymidine). Culture iscontinued in the above medium using the HAT medium for a period of timesufficient to kill cells other than the desired hybridomas (non-fusedcells). Typically, the period is several days to several weeks. Then,hybridomas producing the desired antibody are screened and singly clonedby conventional limiting dilution methods.

The hybridomas thus obtained can be selected using a selection mediumbased on the selection marker possessed by the myeloma used for cellfusion. For example, HGPRT- or TK-deficient cells can be selected byculture using the HAT medium (a culture medium containing hypoxanthine,aminopterin, and thymidine). Specifically, when HAT-sensitive myelomacells are used for cell fusion, cells successfully fused with normalcells can selectively proliferate in the HAT medium. Culture iscontinued in the above medium using the HAT medium for a period of timesufficient to kill cells other than the desired hybridomas (non-fusedcells). Specifically, desired hybridomas can be selected by culture forgenerally several days to several weeks. Then, hybridomas producing thedesired antibody are screened and singly cloned by conventional limitingdilution methods.

Screening and single cloning of desired antibodies can be suitablyperformed by screening methods based on known antigen-antibody reaction.For example, a desired antibody can be selected by screening usingfluorescence activated cell sorting (FACS). FACS is a system thatenables measurement of the binding of an antibody to cell surface byanalyzing cells contacted with a fluorescent antibody using laser beam,and measuring the fluorescence emitted from individual cells.

To screen for hybridomas that produce a monoclonal antibody of thepresent invention by FACS, cells that express the antigen bound by theproduced antibody are first prepared. Preferred cells used for screeningare mammalian cells that are forced to express the antigen. By usingmammalian cells that are used as the host cell but have not beentransformed as a control, the activity of an antibody to bind to thecell-surface antigen can be selectively detected. Specifically,hybridomas producing a desired monoclonal antibody can be obtained byselecting hybridomas that produce an antibody which binds to cellsforced to express the antigen but not to the host cell.

Alternatively, cells expressing the antigen of interest are immobilizedand the activity of an antibody to bind to the antigen-expressing cellscan be assessed based on the principle of ELISA. For example,antigen-expressing cells are immobilized to the wells of an ELISA plate.Culture supernatants of hybridomas are contacted with the immobilizedcells in the wells, and antibodies that bind to the immobilized cellsare detected. When the monoclonal antibodies are derived from mouse,antibodies bound to the cells can be detected using an anti-mouseimmunoglobulin antibody. Hybridomas producing a desired antibody havingthe antigen-binding ability are selected by the above screening, andthey can be cloned by a limiting dilution method or the like.

Monoclonal antibody-producing hybridomas thus prepared can be passagedin a conventional culture medium. The hybridomas can be stored in liquidnitrogen for a long period.

The above hybridomas are cultured by a conventional method, and desiredmonoclonal antibodies can be obtained from the culture supernatants.Alternatively, the hybridomas are administered to and grown incompatible mammals, and monoclonal antibodies can be obtained from theascites. The former method is suitable for obtaining antibodies withhigh purity.

Antibodies that are encoded by antibody genes cloned fromantibody-producing cells such as the above hybridomas can also bepreferably used. A cloned antibody gene is inserted into an appropriatevector, and this is introduced into a host to express the antibodyencoded by the gene. Methods for isolating antibody genes, inserting thegenes into vectors, and transforming host cells have already beenestablished, for example, by Vandamme et al. (Eur. J. Biochem. (1990)192(3), 767-775). Methods for producing recombinant antibodies are alsoknown as described below.

Generally, to obtain a cDNA encoding the antibody variable region (Vregion), total RNA is first extracted from hybridomas. For example, thefollowing methods can be used as methods for extracting mRNAs fromcells:

-   -   the guanidine ultracentrifugation method (Biochemistry (1979)        18(24), 5294-5299), and    -   the AGPC method (Anal. Biochem. (1987) 162(1), 156-159).

Extracted mRNAs can be purified using the mRNA Purification Kit (GEHealthcare Bioscience) or such. Alternatively, kits for extracting totalmRNA directly from cells, such as the QuickPrep mRNA Purification Kit(GE Healthcare Bioscience), are also commercially available. mRNAs canbe prepared from hybridomas using such kits. cDNAs encoding the antibodyV region can be synthesized from the prepared mRNAs using a reversetranscriptase. cDNAs can be synthesized using the AMV ReverseTranscriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) orsuch. Furthermore, the SMART RACE cDNA amplification kit (Clontech) andthe PCR-based 5′-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23),8998-9002; Nucleic Acids Res. (1989) 17(8), 2919-2932) can beappropriately used to synthesize and amplify cDNAs. In such a cDNAsynthesis process, appropriate restriction enzyme sites described belowmay be introduced into both ends of a cDNA.

The cDNA fragment of interest is purified from the resulting PCRproduct, and then this is ligated to a vector DNA. A recombinant vectoris thus constructed, and introduced into E. coli or such. After colonyselection, the desired recombinant vector can be prepared from thecolony-forming E. coli. Then, whether the recombinant vector has thecDNA nucleotide sequence of interest is tested by a known method such asthe dideoxy nucleotide chain termination method.

The 5′-RACE method which uses primers to amplify the variable regiongene is conveniently used for isolating the gene encoding the variableregion. First, a 5′-RACE cDNA library is constructed by cDNA synthesisusing RNAs extracted from hybridoma cells as a template. A commerciallyavailable kit such as the SMART RACE cDNA amplification kit isappropriately used to synthesize the 5′-RACE cDNA library.

The antibody gene is amplified by PCR using the prepared 5′-RACE cDNAlibrary as a template. Primers for amplifying the mouse antibody genecan be designed based on known antibody gene sequences. The nucleotidesequences of the primers vary depending on the immunoglobulin subclass.Therefore, it is preferable that the subclass is determined in advanceusing a commercially available kit such as the Iso Strip mousemonoclonal antibody isotyping kit (Roche Diagnostics).

Specifically, for example, primers that allow amplification of genesencoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains areused to isolate mouse IgG-encoding genes. In general, a primer thatanneals to a constant region site close to the variable region is usedas a 3′-side primer to amplify an IgG variable region gene. Meanwhile, aprimer attached to a 5′ RACE cDNA library construction kit is used as a5′-side primer.

Immunoglobulins composed of a combination of heavy and light chains maybe reshaped using the thus amplified PCR products. A desired antibodycan be selected by screening using the antigen-binding activity of areshaped immunoglobulin as an indicator.

The screening can be carried out, for example, by the following steps:

-   -   (1) contacting a desired antigen-expressing cell with an        antibody comprising the V region encoded by a cDNA obtained from        a hybridoma;    -   (2) detecting the binding of the antibody to the        antigen-expressing cell; and    -   (3) selecting an antibody that binds to the antigen-expressing        cell.

Methods for detecting the binding of an antibody to theantigen-expressing cells are known. Specifically, the binding of anantibody to the antigen-expressing cells can be detected by theabove-described techniques such as FACS. Fixed samples of theantigen-expressing cells may be appropriately used to assess the bindingactivity of an antibody.

For antibody screening methods that use the binding activity as anindicator, panning methods that use phage vectors can also be usedsuitably. Screening methods using phage vectors are advantageous whenthe antibody genes are obtained from a polyclonal antibody-expressingcell population as heavy-chain and light-chain subclass libraries. Genesencoding the heavy-chain and light-chain variable regions can be linkedby an appropriate linker sequence to form a single-chain Fv (scFv).Phages expressing scFv on their surface can be produced by inserting anscFv-encoding gene into a phage vector. The phages are contacted with anantigen of interest. Then, a DNA encoding scFv having the bindingactivity of interest can be isolated by collecting phages bound to theantigen. This process can be repeated as necessary to enrich scFv havingthe binding activity of interest.

After isolation of the cDNA encoding the V region of the antibody ofinterest, the cDNA is digested with restriction enzymes that recognizethe restriction sites introduced into both ends of the cDNA. Preferredrestriction enzymes recognize and cleave a nucleotide sequence thatoccurs in the nucleotide sequence of the antibody gene at a lowfrequency. Furthermore, a restriction site for an enzyme that produces asticky end is preferably introduced into a vector to insert asingle-copy digested fragment in the correct orientation. The cDNAencoding the V region of the antibody is digested as described above,and this is inserted into an appropriate expression vector to constructan antibody expression vector. In this case, if a gene encoding theantibody constant region (C region) and a gene encoding the above Vregion are fused in-frame, a chimeric antibody is obtained. Herein, a“chimeric antibody” means that the origin of the constant region isdifferent from that of the variable region. Thus, in addition tomouse/human heterochimeric antibodies, human/human allochimericantibodies are included in the chimeric antibodies of the presentinvention. A chimeric antibody expression vector can be constructed byinserting the above V region gene into an expression vector that alreadyhas the constant region. Specifically, for example, a recognitionsequence for a restriction enzyme that excises the above V region genecan be appropriately placed on the 5′ side of an expression vectorcarrying a DNA that encodes a desired antibody constant region (Cregion). A chimeric antibody expression vector is constructed by fusingin-frame two genes digested with the same combination of restrictionenzymes.

To produce a monoclonal antibody, antibody genes are inserted into anexpression vector so that the genes are expressed under the control ofan expression regulatory region. The expression regulatory region forantibody expression includes, for example, enhancers and promoters.Furthermore, an appropriate signal sequence may be attached to the aminoterminus so that the expressed antibody is secreted to the outside ofcells. The signal sequence is cleaved from the carboxyl terminus of theexpressed polypeptide, and the resulting antibody can be secreted to theoutside of cells. Then, appropriate host cells are transformed with theexpression vector, and recombinant cells expressing theantibody-encoding DNA can be obtained.

DNAs encoding the antibody heavy chain (H chain) and light chain (Lchain) are separately inserted into different expression vectors toexpress the antibody gene. An antibody molecule having the H and Lchains can be expressed by co-transfecting the same host cell withvectors inserted with the H chain and L chain. Alternatively, host cellscan be transformed with a single expression vector into which DNAsencoding the H and L chains are inserted (see WO 94/11523).

There are many known combinations of host cells and expression vectorsfor antibody preparation by introducing isolated antibody genes intoappropriate hosts. All these expression systems are applicable toisolation of domains that bind to a molecule expressed on the surface ofa cell having immune response-suppressing function, T cell receptorcomplex-binding domains, cancer-specific antigen-binding domains, tumornecrosis factor (TNF) superfamily-binding domains, and tumor necrosisfactor (TNF) receptor superfamily-binding domains of the presentinvention.

Appropriate eukaryotic cells used as host cells include animal cells,plant cells, and fungal cells. Specifically, the animal cells include,for example, the following cells.

-   -   (1) mammalian cells: CHO, COS, myeloma, baby hamster kidney        (BHK), HeLa, Vero, or such;    -   (2) amphibian cells: Xenopus oocytes, or such; and    -   (3) insect cells: sf9, sf21, Tn5, or such.

In addition, as a plant cell, an antibody gene expression system usingcells derived from the Nicotiana genus such as Nicotiana tabacum isknown. Callus cultured cells can be appropriately used to transformplant cells.

Furthermore, the following cells can be used as fungal cells:

yeasts: the Saccharomyces genus such as Saccharomyces cerevisiae, andthe Pichia genus such as Pichia pastoris; and filamentous fungi: theAspergillus genus such as Aspergillus niger.

Furthermore, antibody gene expression systems that utilize prokaryoticcells are also known. For example, when using bacterial cells, E. colicells, Bacillus subtilis cells, and such can suitably be utilized in thepresent invention. Expression vectors carrying the antibody genes ofinterest are introduced into these cells by transfection. Thetransfected cells are cultured in vitro, and the desired antibody can beprepared from the culture of transformed cells.

In addition to the above-described host cells, transgenic animals canalso be used to produce a recombinant antibody. That is, the antibodycan be obtained from an animal into which the gene encoding the antibodyof interest is introduced. For example, the antibody gene can beconstructed as a fusion gene by inserting in frame into a gene thatencodes a protein produced specifically in milk. Goat f3-casein or suchcan be used, for example, as the protein secreted in milk. DNA fragmentscontaining the fused gene inserted with the antibody gene is injectedinto a goat embryo, and then this embryo is introduced into a femalegoat. Desired antibodies can be obtained as a protein fused with themilk protein from milk produced by the transgenic goat born from theembryo-recipient goat (or progeny thereof). In addition, to increase thevolume of milk containing the desired antibody produced by thetransgenic goat, hormones can be administered to the transgenic goat asnecessary (Bio/Technology (1994) 12 (7), 699-702).

In several embodiments, when an antigen-binding molecule describedherein is administered to human, an antigen-binding domain derived froma genetically recombinant antibody that has been artificially modifiedto reduce the heterologous antigenicity against human and such, can beappropriately used as the various binding domains in the molecule whendomains comprising an antibody variable region are used. Suchgenetically recombinant antibodies include, for example, humanizedantibodies. These modified antibodies are appropriately produced byknown methods.

In several embodiments, an antibody variable region used to produce thevarious binding domains of antigen-binding molecules described herein isgenerally formed by three complementarity-determining regions (CDRs)that are separated by four framework regions (FRs). CDR is a region thatsubstantially determines the binding specificity of an antibody. Theamino acid sequences of CDRs are highly diverse. On the other hand, theFR-forming amino acid sequences often have high identity even amongantibodies with different binding specificities. Therefore, generally,the binding specificity of a certain antibody can be introduced intoanother antibody by CDR grafting.

A humanized antibody is also called a reshaped human antibody.Specifically, humanized antibodies prepared by grafting the CDR of anon-human animal antibody such as a mouse antibody to a human antibodyand such are known. Common genetic engineering techniques for obtaininghumanized antibodies are also known. Specifically, for example, overlapextension PCR is known as a method for grafting a mouse antibody CDR toa human FR. In overlap extension PCR, a nucleotide sequence encoding amouse antibody CDR to be grafted is added to primers for synthesizing ahuman antibody FR. Primers are prepared for each of the four FRs. It isgenerally considered that when grafting a mouse CDR to a human FR,selecting a human FR that has high identity to a mouse FR isadvantageous for maintaining the CDR function. That is, it is generallypreferable to use a human FR comprising an amino acid sequence which hashigh identity to the amino acid sequence of the FR adjacent to the mouseCDR to be grafted.

Nucleotide sequences to be ligated are designed so that they will beconnected to each other in frame. Human FRs are individually synthesizedusing the respective primers. As a result, products in which the mouseCDR-encoding DNA is attached to the individual FR-encoding DNAs areobtained. Nucleotide sequences encoding the mouse CDR of each productare designed so that they overlap with each other. Then, complementarystrand synthesis reaction is conducted to anneal the overlapping CDRregions of the products synthesized using a human antibody gene astemplate. Human FRs are ligated via the mouse CDR sequences by thisreaction.

The full length V region gene, in which three CDRs and four FRs areultimately ligated, is amplified using primers that anneal to its 5′- or3′-end, which are added with suitable restriction enzyme recognitionsequences. An expression vector for humanized antibody can be producedby inserting the DNA obtained as described above and a DNA that encodesa human antibody C region into an expression vector so that they willligate in frame. After the recombinant vector is transfected into a hostto establish recombinant cells, the recombinant cells are cultured, andthe DNA encoding the humanized antibody is expressed to produce thehumanized antibody in the cell culture (see, European Patent PublicationNo. EP 239400 and International Patent Publication No. WO 1996/002576).

By qualitatively or quantitatively measuring and evaluating theantigen-binding activity of the humanized antibody produced as describedabove, one can suitably select human antibody FRs that allow CDRs toform a favorable antigen-binding site when ligated through the CDRs.Amino acid residues in FRs may be substituted as necessary, so that theCDRs of a reshaped human antibody form an appropriate antigen-bindingsite. For example, amino acid sequence mutations can be introduced intoFRs by applying the PCR method used for grafting a mouse CDR into ahuman FR. More specifically, partial nucleotide sequence mutations canbe introduced into primers that anneal to the FR Nucleotide sequencemutations are introduced into the FRs synthesized by using such primers.Mutant FR sequences having the desired characteristics can be selectedby measuring and evaluating the activity of the amino acid-substitutedmutant antibody to bind to the antigen by the above-mentioned method(Sato, K. et al., Cancer Res. (1993) 53: 851-856).

Alternatively, desired human antibodies can be obtained by immunizingtransgenic animals having the entire repertoire of human antibody genes(see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO1996/034096; WO 1996/033735) by DNA immunization.

Furthermore, techniques for preparing human antibodies by panning usinghuman antibody libraries are also known. For example, the V region of ahuman antibody is expressed as a single-chain antibody (scFv) on phagesurface by the phage display method. Phages expressing an scFv thatbinds to the antigen can be selected. The DNA sequence encoding thehuman antibody V region that binds to the antigen can be determined byanalyzing the genes of selected phages. The DNA sequence of the scFvthat binds to the antigen is determined. An expression vector isprepared by fusing the V region sequence in frame with the C regionsequence of a desired human antibody, and inserting this into anappropriate expression vector. The expression vector is introduced intocells appropriate for expression such as those described above. Thehuman antibody can be produced by expressing the human antibody-encodinggene in the cells. These methods are already known (see WO 1992/001047;WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO1995/001438; WO 1995/015388).

In addition to the phage display method, techniques that use a cell-freetranslation system, techniques for displaying antigen-binding moleculeson the surface of viruses or cells, and techniques that use emulsionsare also known as techniques for obtaining human antibodies by panningusing human antibody libraries. For example, the ribosome display methodwhere a complex is formed between the translated protein and mRNA viathe ribosome by removing the stop codon and such, the cDNA displaymethod or the mRNA display method where a genetic sequence and thetranslated protein are covalently linked using a compound such aspuromycin, the CIS display method where a complex is formed between thegene and the translated protein using a nucleic acid-binding protein, orsuch may be used as techniques of using a cell-free translation system.For the technique of presenting antigen-binding molecules on the surfaceof cells or viruses, besides the phage display method, the E. colidisplay method, Gram-positive bacteria display method, yeast displaymethod, mammalian cell display method, virus display method, and suchmay be used. As a technique that uses emulsions, the in vitro virusdisplay method which involves incorporating genes andtranslation-related molecules into an emulsion, and such may be used.These methods are already publicly known (Nat Biotechnol. 2000 December;18(12): 1287-92; Nucleic Acids Res. 2006; 34(19): e127; Proc Natl AcadSci USA. 2004 Mar. 2; 101(9):2806-10; Proc Natl Acad Sci USA. 2004 Jun.22; 101(25):9193-8; Protein Eng Des Sel. 2008 April; 21(4):247-55; ProcNatl Acad Sci USA. 2000 Sep. 26; 97(20):10701-5; MAbs. 2010September-October; 2(5):508-18; and Methods Mol Biol. 2012, 911:183-98).

Specific

In several embodiments, “specific” according to the present inventionmeans a condition where one of the molecules involved in specificbinding does not show any significant binding to molecules other than asingle or a number of binding partner molecules. Furthermore, “specific”is also used when an antigen-binding domain is specific to a particularepitope among multiple epitopes contained in an antigen. When an epitopebound by an antigen-binding domain is contained in multiple differentantigens, antigen-binding molecules containing the antigen-bindingdomain can bind to various antigens that have the epitope.

Epitopes

In several embodiments, an “epitope” means an antigenic determinant inan antigen, and refers to an antigen site to which various bindingdomains in antigen-binding molecules disclosed herein bind. Thus, forexample, an epitope can be defined according to its structure.Alternatively, the epitope may be defined according to theantigen-binding activity of an antigen-binding molecule that recognizesthe epitope. When the antigen is a peptide or polypeptide, the epitopecan be specified by the amino acid residues that form the epitope.Alternatively, when the epitope is a sugar chain, the epitope can bespecified by its specific sugar chain structure.

A linear epitope is an epitope that contains an epitope whose primaryamino acid sequence is recognized. Such a linear epitope typicallycontains at least three and most commonly at least five, for example,about 8 to 10 or 6 to 20 amino acids in its specific sequence.

In contrast to the linear epitope, “conformational epitope” is anepitope in which the primary amino acid sequence containing the epitopeis not the only determinant of the recognized epitope (for example, theprimary amino acid sequence of a conformational epitope is notnecessarily recognized by an epitope-defining antibody). Conformationalepitopes may contain a greater number of amino acids compared to linearepitopes. A conformational epitope-recognizing antibody recognizes thethree-dimensional structure of a peptide or protein. For example, when aprotein molecule folds and forms a three-dimensional structure, aminoacids and/or polypeptide main chains that form a conformational epitopebecome aligned, and the epitope is made recognizable by the antibody.Methods for determining epitope conformations include, for example, Xray crystallography, two-dimensional nuclear magnetic resonancespectroscopy, site-specific spin labeling, and electron paramagneticresonance spectroscopy, but are not limited thereto. See, for example,Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol.66, Morris (ed.).

Examples of a method for assessing the binding of an epitope in amolecule expressed on the surface of a cell having immuneresponse-suppressing function by a test antigen-binding molecule areshown below. According to the examples below, methods for assessing thebinding of an epitope in a target antigen by another binding domain, forexample, a T cell receptor complex-binding domain, a cancer-specificantigen-binding domain, a tumor necrosis factor (TNF)superfamily-binding domain, or a tumor necrosis factor (TNF) receptorsuperfamily-binding domain, can also be appropriately conducted.

For example, when CTLA4 is selected as the molecule expressed on thesurface of a cell having immune response-suppressing function, whether atest antigen-binding molecule that comprises an antigen-binding domainfor the molecule recognizes a linear epitope in the antigen molecule canbe confirmed for example as mentioned below. For example, a linearpeptide comprising an amino acid sequence forming the extracellulardomain of CTLA4 is synthesized for the above purpose. The peptide can besynthesized chemically, or obtained by genetic engineering techniquesusing a region in a cDNA of CTLA4 encoding the amino acid sequence thatcorresponds to the extracellular domain. Then, a test antigen-bindingmolecule containing an antigen-binding domain for CTLA4 is assessed forits binding activity towards a linear peptide comprising theextracellular domain-constituting amino acid sequence. For example, animmobilized linear peptide can be used as an antigen to evaluate thebinding activity of the antigen-binding molecule towards the peptide byELISA. Alternatively, the binding activity towards a linear peptide canbe assessed based on the level at which the linear peptide inhibitsbinding of the antigen-binding molecule to CTLA4-expressing cells. Thebinding activity of the antigen-binding molecule towards the linearpeptide can be demonstrated by these tests.

Whether the above-mentioned test antigen-binding molecule containing anantigen-binding domain towards an antigen recognizes a conformationalepitope can be confirmed as below. For example, the above-mentionedantigen-binding molecule that comprises an antigen-binding domain forCTLA4 strongly binds to CTLA4-expressing cells upon contact, but doesnot substantially bind to an immobilized linear peptide comprising anamino acid sequence forming the extracellular domain of CTLA4. Herein,“does not substantially bind” means that the binding activity is 80% orless, generally 50% or less, preferably 30% or less, and particularlypreferably 15% or less compared to the binding activity toantigen-expressing cells.

Methods for assaying the binding activity of a test antigen-bindingmolecule comprising an antigen-binding domain to antigen-expressingcells include, for example, the methods described in Antibodies ALaboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory(1988) 359-420). Specifically, the assessment can be performed based onthe principle of ELISA or fluorescence activated cell sorting (FACS)using antigen-expressing cells as antigen.

In the ELISA format, the binding activity of a test antigen-bindingmolecule comprising an antigen-binding domain towards antigen-expressingcells can be assessed quantitatively by comparing the levels of signalsgenerated by enzymatic reaction. Specifically, a test antigen-bindingmolecule is added to an ELISA plate onto which antigen-expressing cellsare immobilized. Then, the test antigen-binding molecule bound to thecells is detected using an enzyme-labeled antibody that recognizes thetest antigen-binding molecule. Alternatively, when FACS is used, adilution series of a test antigen-binding molecule is prepared, and theantibody-binding titer for antigen-expressing cells can be determined tocompare the binding activity of the test antigen-binding moleculetowards antigen-expressing cells.

The binding of a test antigen-binding molecule to an antigen expressedon the surface of cells suspended in buffer or the like can be detectedusing a flow cytometer. Known flow cytometers include, for example, thefollowing devices:

-   -   FACSCanto™ II    -   FACSAria™    -   FACSArray™    -   FACSVantage™ SE    -   FACSCalibur™ (all are trade names of BD Biosciences)    -   EPICS ALTRA HyPerSort    -   Cytomics FC 500    -   EPICS XL-MCL ADC EPICS XL ADC    -   Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of        Beckman Coulter).

Suitable methods for assaying the binding activity of theabove-mentioned test antigen-binding molecule comprising anantigen-binding domain towards an antigen include, for example, themethod below. First, antigen-expressing cells are reacted with a testantigen-binding molecule, and then this is stained with an FITC-labeledsecondary antibody that recognizes the antigen-binding molecule. Thetest antigen-binding molecule is appropriately diluted with a suitablebuffer to prepare and use it at a desired concentration. For example,the molecule can be used at a concentration within the range of 10 μg/mlto 10 ng/ml. Then, the fluorescence intensity and cell count aredetermined using FACSCalibur (BD). The fluorescence intensity obtainedby analysis using the CELL QUEST Software (BD), i.e., the Geometric Meanvalue, reflects the quantity of antibody bound to the cells. That is,the binding activity of a test antigen-binding molecule, which isrepresented by the quantity of the test antigen-binding molecule bound,can be measured by determining the Geometric Mean value.

In several embodiments, whether a test antigen-binding moleculecomprising an antigen-binding domain of the present invention shares acommon epitope with another antigen-binding molecule can be assessedbased on competition between the two molecules for the same epitope. Thecompetition between antigen-binding molecules can be detected by across-blocking assay or the like. For example, the competitive ELISAassay is a preferred cross-blocking assay.

Specifically, in a cross-blocking assay, the antigen coating the wellsof a microtiter plate is pre-incubated in the presence or absence of acandidate competitor antigen-binding molecule, and then a testantigen-binding molecule is added thereto. The quantity of testantigen-binding molecule bound to the antigen in the wells indirectlycorrelates with the binding ability of a candidate competitorantigen-binding molecule that competes for the binding to the sameepitope. That is, the greater the affinity of the competitorantigen-binding molecule for the same epitope, the lower the bindingactivity of the test antigen-binding molecule towards the antigen-coatedwells.

The quantity of the test antigen-binding molecule bound to the wells viathe antigen can be readily determined by labeling the antigen-bindingmolecule in advance. For example, a biotin-labeled antigen-bindingmolecule can be measured using an avidin/peroxidase conjugate andappropriate substrate. In particular, a cross-blocking assay that usesenzyme labels such as peroxidase is called “competitive ELISA assay”.The antigen-binding molecule can also be labeled with other labelingsubstances that enable detection or measurement. Specifically,radiolabels, fluorescent labels, and such are known.

When the candidate competitor antigen-binding molecule can block thebinding of a test antigen-binding molecule comprising an antigen-bindingdomain by at least 20%, preferably at least 20 to 50%, and morepreferably at least 50% compared to the binding activity in a controlexperiment conducted in the absence of the competitor antigen-bindingmolecule, the test antigen-binding molecule is determined tosubstantially bind to the same epitope bound by the competitorantigen-binding molecule, or to compete for binding to the same epitope.

In several embodiments, when the structure of an epitope bound by a testantigen-binding molecule comprising an antigen-binding domain of thepresent invention is already identified, whether the test and controlantigen-binding molecules share a common epitope can be assessed bycomparing the binding activities of the two antigen-binding moleculestowards a peptide prepared by introducing amino acid mutations into thepeptide forming the epitope.

As a method for measuring such binding activities, for example, thebinding activities of test and control antigen-binding molecules towardsa linear peptide into which a mutation is introduced are measured bycomparison in the above ELISA format. Besides the ELISA methods, thebinding activity towards the mutant peptide bound to a column can bedetermined by passing the test and control antigen-binding moleculesthrough the column, and then quantifying the antigen-binding moleculeeluted in the eluate. Methods for adsorbing a mutant peptide to acolumn, for example, in the form of a GST fusion peptide, are known.

Alternatively, when the identified epitope is a conformational epitope,whether test and control antigen-binding molecules share a commonepitope can be assessed by the following method. First, cells expressingan antigen which is a binding target of an antigen-binding domain andcells expressing an antigen having an epitope introduced with a mutationare prepared. The test and control antigen-binding molecules are addedto a cell suspension prepared by suspending these cells in anappropriate buffer such as PBS. Then, the cell suspension isappropriately washed with a buffer, and an FITC-labeled antibody thatcan recognize the test and control antigen-binding molecules is addedthereto. The fluorescence intensity and number of cells stained with thelabeled antibody are determined using FACSCalibur (BD). The test andcontrol antigen-binding molecules are appropriately diluted using asuitable buffer, and used at desired concentrations. For example, theymay be used at a concentration within the range of 10 μg/ml to 10 ng/ml.The fluorescence intensity determined by analysis using the CELL QUESTSoftware (BD), i.e., the Geometric Mean value, reflects the quantity ofthe labeled antibody bound to the cells. That is, the binding activitiesof the test and control antigen-binding molecules, which are representedby the quantity of the labeled antibody bound, can be measured bydetermining the Geometric Mean value.

In several embodiments, an “antigen-binding molecule” of the presentinvention comprises both heavy and light chains which form an “antibodyvariable region” of this invention within a single polypeptide chain;however, it may be an antibody fragment lacking a constant region.Examples of such antibody fragments include a diabody (Db), an scFv, asingle-chain antibody, an sc(Fv)2, an sc(Fab′)2, Fab, F(ab′)2, and Fab′.

Db is a dimer composed of two polypeptide chains (Holliger P et al.,Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; WO93/11161).In each polypeptide chain, an L-chain variable region (VL) and anH-chain variable region (VH) are linked by a linker short enough so thatthese two regions on the same chain cannot associate with each other,for example, a linker of about five residues.

Because the linker between VL and VH is too short for formation of asingle chain variable region fragment, VL and VH encoded on the samepolypeptide chain dimerize to form two antigen-binding sites.

scFv, Single-Chain Antibody, and Sc(Fv)2

Furthermore, in several embodiments, the terms “scFv”, “single-chainantibody”, and “sc(Fv)2” described herein all refer to an antibodyfragment of a single polypeptide chain that contains variable regionsderived from the heavy and light chains, but not the constant region. Ingeneral, a single-chain antibody also contains a polypeptide linkerbetween the VH and VL domains, which enables formation of a desiredstructure that is thought to allow antigen binding. The single-chainantibody is discussed in detail by Pluckthun in “The Pharmacology ofMonoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds.,Springer-Verlag, New York, 269-315 (1994)”. See also InternationalPatent Publication WO 1988/001649; U.S. Pat. Nos. 4,946,778 and5,260,203. In a particular embodiment, the single-chain antibody can bebispecific and/or humanized.

scFv is an antigen-binding domain in which VH and VL forming Fv arelinked together by a peptide linker (Proc. Natl. Acad. Sci. U.S.A.(1988) 85(16), 5879-5883). VH and VL can be retained in close proximityby the peptide linker.

sc(Fv)2 is a single-chain antibody in which four variable regions of twoVL and two VH are linked by linkers such as peptide linkers to form asingle chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VHand two VL may be derived from different monoclonal antibodies. Suchsc(Fv)2 preferably includes, for example, a bispecific sc(Fv)2 thatrecognizes two types of epitopes present in a single antigen asdisclosed in the Journal of Immunology (1994) 152(11), 5368-5374.sc(Fv)2 can be produced by methods known to those skilled in the art.For example, sc(Fv)2 can be produced by linking scFv by a linker such asa peptide linker.

In several embodiments, the form of an antigen-binding domain forming ansc(Fv)2 described herein include an antibody in which the two VH unitsand two VL units are arranged in the order of VH, VL, VH, and VL([VH]-linker-[VL]-linker-[VH]-linker-[VL]) beginning from the N terminusof a single-chain polypeptide. The order of the two VH units and two VLunits is not limited to the above form, and they may be arranged in anyorder. Example order of the form is listed below.

-   -   [VL]-linker-[VH]-linker-[VH]-linker-[VL]    -   [VH]-linker-[VL]-linker-[VL]-linker-[VH]    -   [VH]-linker-[VH]-linker-[VL]-linker-[VL]    -   [VL]-linker-[VL]-linker-[VH]-linker-[VH]    -   [VL]-linker-[VH]-linker-[VL]-linker-[VH]

The molecular form of sc(Fv)2 is also described in detail inWO2006/132352. According to these descriptions, those skilled in the artcan appropriately prepare desired sc(Fv)2 to produce the antigen-bindingmolecules disclosed herein.

Herein, the term “variable fragment (Fv)” refers to the minimum unit ofan antibody-derived antigen-binding domain composed of a pair of theantibody light chain variable region (VL) and antibody heavy chainvariable region (VH). In 1988, Skerra and Pluckthun found thathomogeneous and active antibodies can be prepared from the E. coliperiplasm fraction by inserting an antibody gene downstream of abacterial signal sequence and inducing expression of the gene in E. coli(Science (1988) 240(4855), 1038-1041). In the Fv prepared from theperiplasm fraction, VH associates with VL in a manner so as to bind toan antigen.

Furthermore, in several embodiments, the antigen-binding molecule of thepresent invention may be conjugated with a carrier polymer such as PEGor an organic compound such as an anticancer agent. Alternatively, aglycosylation sequence can be inserted to suitably add a sugar chain forthe purpose of producing a desired effect.

The linkers to be used for linking the variable regions of an antibodycomprise arbitrary peptide linkers that can be introduced by geneticengineering, or synthetic linkers (for example, linkers disclosed inProtein Engineering, 9(3), 299-305, 1996). However, peptide linkers arepreferred in the present invention. The length of the peptide linkers isnot particularly limited, and can be suitably selected by those skilledin the art according to the purpose. The length is preferably five aminoacids or more (without particular limitation, the upper limit isgenerally 30 amino acids or less, preferably 20 amino acids or less),and particularly preferably 15 amino acids. When sc(Fv)2 contains threepeptide linkers, their length may be all the same or different.

For example, such peptide linkers include:

-   -   Ser    -   Gly⋅Ser    -   Gly⋅Gly⋅Ser    -   Ser⋅Gly⋅Gly    -   Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 20)    -   Ser⋅Gly⋅Gly⋅Gly (SEQ ID NO: 21)    -   Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 22)    -   Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 23)    -   Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 24)    -   Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 25)    -   Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 26)    -   Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 27)    -   (Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 22))n    -   (Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 23))n        where n is an integer of 1 or larger. The length or sequences of        peptide linkers can be selected accordingly by those skilled in        the art depending on the purpose.

Synthetic linkers (chemical crosslinking agents) is routinely used tocrosslink peptides, and for example:

-   -   N-hydroxy succinimide (NHS),    -   disuccinimidyl suberate (DSS),    -   bis(sulfosuccinimidyl) suberate (BS3),    -   dithiobis(succinimidyl propionate) (DSP),    -   dithiobis(sulfosuccinimidyl propionate) (DTSSP),    -   ethylene glycol bis(succinimidyl succinate) (EGS),    -   ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS),    -   disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate        (sulfo-DST),    -   bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES),    -   and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone        (sulfo-BSOCOES).    -   These crosslinking agents are commercially available.

In general, three linkers are required to link four antibody variableregions together. The linkers to be used may be of the same type ordifferent types.

Fab. F(ab′)2. and Fab′

Furthermore, “Fab” is composed of a single light chain, and a CH1 domainand variable region from a single heavy chain. The heavy chain of Fabmolecule cannot form disulfide bonds with another heavy chain molecule.

“F(ab′)2” or “Fab′” is produced by treating an immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andrefers to an antibody fragment generated by digesting an immunoglobulin(monoclonal antibody) at near the disulfide bonds present between thehinge regions in each of the two H chains. For example, papain cleavesIgG upstream of the disulfide bonds present between the hinge regions ineach of the two H chains to generate two homologous antibody fragments,in which an L chain comprising VL (L-chain variable region) and CL(L-chain constant region) is linked to an H-chain fragment comprising VH(H-chain variable region) and CHγ1 (γ1 region in an H-chain constantregion) via a disulfide bond at their C-terminal regions. Each of thesetwo homologous antibody fragments is called Fab′.

“F(ab′)2” contains two light chains and two heavy chains comprising theconstant region of a CH1 domain and a portion of a CH2 domain so thatdisulfide bonds are formed between the two heavy chains. In severalembodiments, the F(ab′)2 constituting an antigen-binding moleculedisclosed herein can be preferably obtained as below. A full-lengthmonoclonal antibody or such comprising a desired antigen-binding domainis partially digested with a protease such as pepsin, and then Fcfragments are removed by adsorption onto a Protein A column. Theprotease is not particularly limited, as long as it can digest thefull-length antibody in a restrictive manner to produce F(ab′)2 under anappropriately established enzyme reaction condition such as pH. Suchproteases include, for example, pepsin and ficin.

Multispecific Antibodies

A preferred embodiment of the “antigen-binding molecule” or “antibody”of the present invention includes a multispecific antibody. When usingan Fc region with decreased Fcγ receptor-binding activity as the Fcregion of a multispecific antibody, an Fc region derived from amultispecific antibody may also be used appropriately. For themultispecific antibodies of the present invention, in particular,bispecific antibodies are preferred.

For association of multispecific antibodies, one can apply the techniqueof introducing charge repulsion at the interface of the second constantregion of the antibody H chain (CH2) or the third constant region of theH chain (CH3) to suppress undesired associations between H chains(WO2006/106905).

In the technique of suppressing unintended association between H chainsby introducing charge repulsion at the interface of CH2 or CH3, examplesof the amino acid residues that are contacted at the interface of otherconstant regions of the H chain include the region facing the residue atposition 356 (EU numbering), the residue at position 439 (EU numbering),the residue at position 357 (EU numbering), the residue at position 370(EU numbering), the residue at position 399 (EU numbering), and theresidue at position 409 (EU numbering) in the CH3 region.

More specifically, for example, for an antibody comprising two types ofH chain CH3 regions, the antibody can be made so that one to three pairsof amino acid residues selected from the amino acid residue pairs shownbelow in (1) to (3) in the first H chain CH3 region have the samecharge: (1) amino acid residues at positions 356 and 439 (EU numbering)which are amino acid residues contained in the H chain CH3 region; (2)amino acid residues at positions 357 and 370 (EU numbering) which areamino acid residues contained in the H chain CH3 region; and (3) aminoacid residues at positions 399 and 409 (EU numbering) which are aminoacid residues contained in the H chain CH3 region.

Furthermore, the antibody can be made so that one to three pairs ofamino acid residues corresponding to the amino acid residue pairs shownabove in (1) to (3) having the same type of charge in the first H chainCH3 region, which are amino acid residue pairs selected from the aminoacid residue pairs shown above in (1) to (3) in the second H chain CH3region which differs from the first H chain CH3 region, have a chargeopposite to the corresponding amino acid residues in the aforementionedfirst H chain CH3 region.

The respective amino acid residues of(1) to (3) mentioned above arepositioned close to each other when associated. For the desired H chainCH3 region or H chain constant region, those skilled in the art can findsites corresponding to the above-mentioned amino acid residues of(1) to(3) by homology modeling and such using commercially available software,and amino acid residues of these sites can be subjected to modificationsas appropriate.

In the above-mentioned antibodies, “amino acid residues having a charge”are preferably selected, for example, from amino acid residues containedin either one of groups (a) and (b) below:

-   -   (a) glutamic acid (E) and aspartic acid (D); and    -   (b) lysine (K), arginine (R), and histidine (H).

Regarding the above-mentioned antibodies, “having the same type ofcharge” means, for example, that two or more amino acid residues allhave amino acid residues included in either one of the above-mentionedgroups (a) and (b). The phrase “having the opposite charge” means that,for example, when at least one of the two or more amino acid residueshas an amino acid residue included in either one of the above-mentionedgroups (a) and (b), the remaining amino acid residue(s) will have anamino acid residue included in the other group.

In a preferred embodiment of the above-mentioned antibody, the first Hchain CH3 region and the second H chain CH3 region may be cross-linkedby a disulfide bond.

In the present invention, the amino acid residue to be subjected toalteration is not limited to an amino acid residue of the constantregion or variable region of the antibody described above. With regardto polypeptide mutants or heteromultimers, those skilled in the art canfind amino acid residues that form the interface through homologymodeling and such using commercially available software, and can subjectthe amino acid residues at those sites to alterations so thatassociation is regulated.

In several embodiments, other known techniques can also be used for theassociation of multispecific antibodies of the present invention.Polypeptides with different amino acids having an Fc region can beefficiently associated with each other by substituting an amino acidside chain present in one of the H chain variable regions of theantibody with a larger side chain (knob), and substituting an amino acidside chain present in the corresponding variable region of the other Hchain with a smaller side chain (hole), to allow placement of the knobwithin the hole (WO 1996/027011; Ridgway J B et al., Protein Engineering(1996) 9, 617-621; Merchant A M et al. Nature Biotechnology (1998) 16,677-681; and US20130336973).

In addition, other known techniques can also be used to formmultispecific antibodies of the present invention. Association ofpolypeptides having different sequences can be induced efficiently bycomplementary association of CH3s, using a strand-exchange engineeredCH3 domain produced by changing part of CH3 in one of the H chains of anantibody into its corresponding IgA-derived sequence, and introducinginto the complementary portion of the CH3 in the other H chain itscorresponding IgA-derived sequence (Protein Engineering Design &Selection, 23; 195-202, 2010). This known technique can also be used toefficiently form multispecific antibodies of interest.

In addition, the following techniques and such may be used for theformation of multispecific antibodies: techniques for antibodyproduction using association of antibody CH1 and CL, and association ofVH and VL as described in WO 2011/028952, WO2014/018572, and NatBiotechnol. 2014 February; 32(2): 191-8; techniques for producingbispecific antibodies using separately prepared monoclonal antibodies incombination (Fab Arm Exchange) as described in WO2008/119353 andWO2011/131746; techniques for regulating association between antibodyheavy chain CH3s as described in WO2012/058768 and WO2013/063702;techniques for producing bispecific antibodies composed of two types oflight chains and one type of heavy chain as described in WO2012/023053;techniques for producing bispecific antibodies using two bacterial cellstrains that individually express one of the chains of an antibodycomprising a single H chain and a single L chain as described byChristoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)).

An embodiment of multispecific antibody formation includes methods forobtaining bispecific antibodies by mixing two types of monoclonalantibodies in the presence of a reducing agent to cleave the disulfidebonds in the core hinge region, followed by re-association forheterodimerization (FAE) as described above. Meanwhile, introduction ofelectrostatic interactions at the interacting interface of the CH3region (WO2006/106905) can induce even more efficient heterodimerizationduring the re-association (WO2015/046467). In FAE usingnaturally-occurring IgG, re-association takes place randomly; and thustheoretically, bispecific antibodies can only be obtained at 50%efficiency; however, in this method, bispecific antibodies can beproduced in high yield.

Alternatively, even when a multispecific antibody of interest cannot beformed efficiently, a multispecific antibody of the present inventioncan be obtained by separating and purifying the multispecific antibodyof interest from the produced antibodies. For example, a method has beenreported that enables purification of two types of homologous forms andthe heterologous antibody of interest by ion exchange chromatography, byconferring a difference in the isoelectric points by introducing aminoacid substitutions into the variable regions of the two types of Hchains (WO2007114325). To date, as a method for purifying heterologousforms, a method using Protein A to purify a heterodimerized antibodycomprising a mouse IgG2a H chain that binds to Protein A and a rat IgG2bH chain that does not bind to Protein A has been reported (WO98050431and WO95033844). Furthermore, the heterodimerized antibody per se can bepurified efficiently using a Protein A column by changing theinteraction between each of the H chains and Protein A, by using Hchains in which amino acid residues at the IgG-Protein A binding site,positions 435 and 436 (EU numbering), are substituted with amino acidsthat yield a different binding strength to Protein A such as Tyr, His,or such.

Alternatively, a common L chain that can confer binding ability to aplurality of different H chains can be obtained and used as the common Lchain ofa multispecific antibody. Efficient expression of amultispecific IgG can be achieved by introducing the genes of such acommon L chain and a plurality of different H chains into cells andexpressing the IgG (Nature Biotechnology (1998) 16, 677-681). A methodfor selecting a common L chain that shows strong binding ability to anydifferent H chains can also be used when selecting a common H chain (WO2004/065611).

Furthermore, an Fc region whose C-terminal heterogeneity has beenimproved can be appropriately used as an Fc region of the presentinvention. More specifically, Fc regions lacking glycine at position 446and lysine at position 447, as specified by EU numbering, in the aminoacid sequences of two polypeptides constituting an Fc region derivedfrom IgG1, IgG2, IgG3, or IgG4, are provided.

A plurality, such as two or more, of these techniques can be used incombination. Furthermore, these techniques can be appropriately andseparately applied to the two H chains to be associated. Furthermore,these techniques can be used in combination with the above-mentioned Fcregion of which Fcγ receptor-binding activity has been decreased.Furthermore, an antigen-binding molecule of the present invention may bea molecule produced separately based on an antigen-binding moleculesubjected to the above-described modifications so as to have the sameamino acid sequence. T cell-redirecting antigen-binding moleculestowards cells having immunosuppressive functions

In several embodiments, an antigen-binding molecule of the presentinvention is a T cell-redirecting antigen-binding molecule towards cellshaving immunosuppressive functions (T cell-redirecting antigen-bindingmolecule).

In several embodiments, a T cell-redirecting antigen-binding molecule ofthe present invention can be any molecule as long as it comprises:

-   -   (1) a domain that binds to a molecule expressed on the surface        of a cell having an immune response-suppressing function; and    -   (2) a T cell receptor complex-binding domain, and its structure        is not limited. By comprising these two binding domains, the T        cell-redirecting antigen-binding molecule can activate immune        responses through inhibition of the immune response-suppressing        effects of the cell expressing the molecule described in (1),        and induce excellent cytotoxicity on cancer cells or cancer        cell-comprising tumor tissues.    -   The binding domains described in (1) and (2) of the present        invention can be appropriately selected for the above-described        molecules expressed on the surface of the cells having an immune        response-suppressing function, or antigens belonging to the T        cell receptor complex, respectively. These binding domains can        be linked directly by a peptide bond or connected via a linker.

In several embodiments, the T cell-redirecting antigen-binding moleculesof the present invention may further comprise an FcRn-binding domain.When using the Fc region of the above-described antibody as theFcRn-binding domain, an Fc region with decreased Fcγ receptor-bindingactivity is preferred. Reducing the Fcγ receptor-binding activityenables suppression of side effects produced by systemic immuneactivation, such as cytokine release, caused by crosslinking between Fcγreceptor-expressing cells and T cell receptor complex-expressing cells.

In several embodiments, T cell-redirecting antigen-binding molecules ofthe present invention can be produced using the known methods describedabove.

For example, when (i) F(ab′)2 is used as the domain that binds to amolecule expressed on the surface of a cell having immuneresponse-suppressing functions, (ii) F(ab′)2 is used as a T cellreceptor complex-binding domain, and (iii) a domain comprising an Fcregion with decreased Fcγ receptor-binding activity is used as theFcRn-binding domain, and when the antigen-binding domains described in(i) and (ii) and the Fc region-comprising domain described in (iii) aredirectly linked by peptide bonds, the linked polypeptides form anantibody structure. Such antibodies can be produced by purification fromthe above-described hybridoma culture medium, and also by purifyingantibodies from the culture medium of desired host cells that stablycarry polynucleotides encoding polypeptides constituting the antibody.

In addition to the linkers exemplified above, linkers with peptide tagssuch as His tag, HA tag, myc tag, and FLAG tag may also be suitably usedas the linkers to be employed when connecting each of the domains vialinkers. Furthermore, hydrogen bonding, disulfide bonding, covalentbonding, ionic interaction, or the property of mutual binding as aresult of combination thereof may be suitably used. For example, theaffinity between antibody CH1 and CL may be used, and Fc regions derivedfrom the above-described multispecific antibodies may also be used forheterologous Fc region association.

In several embodiments, the present invention also relates topolynucleotides encoding the T cell-redirecting antigen-bindingmolecules of the present invention, and the polynucleotides can beincorporated into arbitrary expression vectors. Suitable hosts can betransformed with the expression vectors to produce cells that expressthe T cell-redirecting antigen-binding molecules. T cell-redirectingantigen-binding molecules encoded by the polynucleotides can be obtainedby culturing cells that express the T cell-redirecting antigen-bindingmolecules, and collecting expression products from the culturesupernatant. That is, the present invention relates to vectorscomprising a polynucleotide that encodes a T cell-redirectingantigen-binding molecule of the present invention, cells carrying such avector, and methods for producing T cell-redirecting antigen-bindingmolecules, which comprise culturing the cells and collecting Tcell-redirecting antigen-binding molecules from the culture supernatant.These can be obtained by techniques similar to those for recombinantantibodies mentioned above. Pharmaceutical compositions comprising Tcell-redirecting antigen-binding molecules towards cells havingimmunosunpressive functions

From another perspective, the present invention provides pharmaceuticalcompositions comprising the above-described T cell-redirectingantigen-binding molecule as an active ingredient. Furthermore, thepresent invention relates to pharmaceutical compositions for inhibitingimmune response-suppressing activity (agents for inhibiting immuneresponse-suppressing activity), agents for activating immune response,agents for inducing cytotoxicity, agents for suppressing cellproliferation (agents for inhibiting cell proliferation), and anticanceragents, each comprising the T cell-redirecting antigen-binding moleculeas an active ingredient (hereinafter, pharmaceutical compositions andsuch). The pharmaceutical compositions can be used as agents fortreating cancer or agents for preventing cancer. The agents forinhibiting immune response-suppressing activity, agents for activatingimmune response, therapeutic agents for inducing cytotoxicity, agentsfor suppressing cell proliferation, and anticancer agents of the presentinvention are preferably administered to individuals suffering fromcancer, or individuals who may relapse.

Furthermore, in the present invention, agents for inhibiting immuneresponse-suppressing activity, agents for activating immune response,therapeutic agents for inducing cytotoxicity, agents for suppressingcell proliferation, and anticancer agents, the agents comprising theabove-described T cell-redirecting antigen-binding molecule as an activeingredient can be presented as methods for inhibiting immuneresponse-suppressing activity, methods for activating immune response,methods for inducing cytotoxicity, methods for suppressing cellproliferation, methods for activating immunity against cancer cells orcancer cell-comprising tumor tissues, or as methods for preventing ortreating cancer, the methods comprising the step of administering the Tcell-redirecting antigen-binding molecule to an individual; or presentedas use of the T cell-redirecting antigen-binding molecules in theproduction of pharmaceutical compositions for inhibiting immuneresponse-suppressing activity, pharmaceutical compositions foractivating immune response, pharmaceutical compositions for inducingcytotoxicity, agents for suppressing cell proliferation, and anticanceragents; or alternatively, presented as T cell-redirectingantigen-binding molecules for use in inhibiting immuneresponse-suppressing activity, activating immune response, inducingcytotoxicity, suppressing cell proliferation, activating immunityagainst cancer cells or cancer cell-comprising tumor tissues, ortreating or preventing cancer.

In the present invention, “comprising a T cell-redirectingantigen-binding molecule as an active ingredient” means containing a Tcell-redirecting antigen-binding molecule as a major active component,and does not limit the content of the T cell-redirecting antigen-bindingmolecule.

Furthermore, pharmaceutical compositions or such comprising a Tcell-redirecting antigen-binding molecule of the present invention canbe used by combining multiple types of T cell-redirectingantigen-binding molecules as necessary. For example, by using a cocktailof a plurality of T cell-redirecting antigen-binding molecules of thepresent invention that bind to the same antigen, one can enhance theeffect on cells expressing the antigen.

If necessary, the T cell-redirecting antigen-binding molecules of thepresent invention may be encapsulated in microcapsules (microcapsulesmade from hydroxymethylcellulose, gelatin, poly[methylmethacrylate], andthe like), and made into components of colloidal drug delivery systems(liposomes, albumin microspheres, microemulsions, nano-particles, andnano-capsules) (for example, see “Remington's Pharmaceutical Science16th edition”, Oslo Ed. (1980)). Moreover, methods for preparing agentsas sustained-release agents are known, and these can be applied to the Tcell-redirecting antigen-binding molecules of the present invention (J.Biomed. Mater. Res. (1981) 15, 267-277; Chemtech. (1982) 12, 98-105;U.S. Pat. No. 3,773,719; European Patent Application (EP) Nos. EP58481and EP 133988; Biopolymers (1983) 22, 547-556).

The pharmaceutical compositions, immune response activating agents,cytotoxicity inducing agents, cell proliferation-suppressing agents(cell proliferation inhibiting agents), or anticancer agents, eachcomprising the T cell-redirecting antigen-binding molecules of thepresent invention may be administered either orally or parenterally topatients. Parental administration is preferred. Specifically, suchadministration methods include injection, nasal administration,transpulmonary administration, and percutaneous administration.Injections include, for example, intravenous injections, intramuscularinjections, intraperitoneal injections, and subcutaneous injections. Forexample, pharmaceutical compositions, immune response activating agents,therapeutic agents for inducing cellular cytotoxicity, cellproliferation-suppressing agents (cell proliferation-inhibiting agents),or anticancer agents, each comprising the T cell-redirectingantigen-binding molecules of the present invention can be administeredlocally or systemically by injection. Furthermore, appropriateadministration methods can be selected according to the patient's ageand symptoms. The administered dose can be selected, for example, fromthe range of 0.0001 mg to 1,000 mg per kg of body weight for eachadministration. Alternatively, the dose can be selected, for example,from the range of 0.001 mg/body to 100,000 mg/body per patient. However,the dose of a pharmaceutical composition or such comprising a Tcell-redirecting antigen-binding molecule of the present invention isnot limited to these doses.

The pharmaceutical compositions or such comprising the Tcell-redirecting antigen-binding molecules of the present invention canbe formulated according to conventional methods (for example,Remington's Pharmaceutical Science, latest edition, Mark PublishingCompany, Easton, U.S.A.), and may also contain pharmaceuticallyacceptable carriers and additives. Examples include, but are not limitedto, surfactants, excipients, coloring agents, flavoring agents,preservatives, stabilizers, buffers, suspension agents, isotonic agents,binders, disintegrants, lubricants, fluidity promoting agents, andcorrigents, and other commonly used carriers can be suitably used.Specific examples of the carriers include light anhydrous silicic acid,lactose, crystalline cellulose, mannitol, starch, carmellose calcium,carmellose sodium, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate,polyvinylpyrrolidone, gelatin, medium-chain triglyceride,polyoxyethylene hardened castor oil 60, saccharose, carboxymethylcellulose, corn starch, inorganic salt, and such. Anticancer agents

In several embodiments, anticancer agents of the present invention arenot particularly limited and any anticancer agent can be used as longas, when used in combination with a T cell-redirecting antigen-bindingmolecule, the therapeutic effect or prophylactic effect of an anticanceragent is enhanced or the therapeutic effect or the prophylactic effectof the T cell-redirecting antigen-binding molecule is enhanced.

In several embodiments, the anticancer agents include, but is notlimited to, nitrogen mustard analogues, alkyl sulfonates, ethyleneimines, nitrosoureas, epoxides, other alkylating agents, folic acidanalogues, purine analogues, pyrimidine analogues, other antimetabolicagents, vinca alkaloids or analogues thereof, podophyllotoxinderivatives, camptothecan analogs, colchicine derivatives, taxanes,other plant alkaloids or natural products, actinomycines, anthracyclinesor related substances, other cytotoxic antibiotics, platinum compounds,methylhydrazines, kinase inhibitors, enzymes, histone deacetylaseinhibitors, retinoids, immune checkpoint inhibitors, monoclonalantibodies and other molecular-targeted drugs, or other anticanceragents.

In several embodiments, an “immune checkpoint” according to the presentinvention refers to a molecule that is expressed on cytotoxic T cellsand binds to a ligand to thereby transduce to the T cells signalsinhibiting the immune response. Examples of the immune checkpointsinclude but are not limited to molecules such as PD-1, CTLA-4, TIM3,LAG3, and VISTA. In several embodiments, an “immune checkpointinhibitor” according to the present invention refers to a pharmaceuticalagent that inhibits binding between an immune checkpoint and its ligand,and thereby inhibits signal transduction mediated by the immunecheckpoint, and examples include PD-1 inhibitors, CTLA-4 inhibitors,TIM3 inhibitors, LAG3 inhibitors, and VISTA inhibitors. An immunecheckpoint inhibitor may be a pharmaceutical agent that binds to theimmune checkpoint, or a pharmaceutical agent that binds to its bindingpartner ligand. For example, a PD-1 inhibitor may be a pharmaceuticalagent that binds to PD-1, or it may be a pharmaceutical agent that bindsto PD-L1 and/or PD-L2, which is PD-1 binding partner ligand(s).

In several embodiments, a PD-1 inhibitor is selected from the groupconsisting of PD-1-binding antagonists, PD-L1-binding antagonists, andPD-L2-binding antagonists.

In several embodiments, a PD-1 inhibitor is a PD-1-binding antagonist.In several embodiments, a PD-1-binding antagonist inhibits binding ofPD-1 to its binding partner ligand. In several embodiments, aPD-1-binding antagonist inhibits binding of PD-1 to PD-L1. In severalembodiments, a PD-1-binding antagonist inhibits binding of PD-1 toPD-L2. In several embodiments, a PD-1-binding antagonist inhibitsbinding of PD-1 to both PD-L1 and PD-L2. In several embodiments, aPD-1-binding antagonist is an antibody. In several embodiments, aPD-1-binding antagonist is MDX-1106 (nivolumab). In several embodiments,a PD-1-binding antagonist is MK-3475 (lambrolizumab). In severalembodiments, a PD-1-binding antagonist is CT-011 (pidilizumab). Inseveral embodiments, a PD-1-binding antagonist is AMP-224.

In several embodiments, a PD-1 inhibitor is a PD-L1-binding antagonist.In several embodiments, a PD-L1-binding antagonist inhibits binding ofPD-L1 to PD-1. In several embodiments, a PD-L1-binding antagonistinhibits binding of PD-L1 to B7-1. In several embodiments, aPD-L1-binding antagonist inhibits binding of PD-L1 to both PD-1 andB7-1. In several embodiments, a PD-L1-binding antagonist is an antibody.In several embodiments, a PD-L1-binding antagonist is selected from thegroup consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. Inseveral embodiments, a PD-L1-binding antagonist is MPDL3280A(atezolizumab). In several embodiments, a PD-L1-binding antagonist isMEDI4736 (durvalumab).

In several embodiments, a PD-1 inhibitor is a PD-L2-binding antagonist.In several embodiments, a PD-L2-binding antagonist is an antibody. Inseveral embodiments, a PD-L2-binding antagonist is immunoadhesin.

Immunoactivating Antigen-Binding Molecules

In several embodiments, an example of anticancer agents of the presentinvention includes an immunoactivating antigen-binding molecule (a firstimmunoactivating antigen-binding molecule). In several embodiments, thefirst immunoactivating antigen-binding molecules can be any molecule aslong as they comprise:

-   -   (1) a cancer-specific antigen-binding domain; and    -   (2) a tumor necrosis factor (TNF) superfamily-binding domain or        a tumor necrosis factor (TNF) receptor superfamily-binding        domain; and their structure is not limited.        By comprising these two binding domains, the immunoactivating        antigen-binding molecules specifically activate cells that        express a molecule belonging to the TNF superfamily or the TNF        receptor superfamily and that express a cancer-specific antigen,        or cells contained in tumor tissues comprising these cells, to        thereby enable induction of excellent (specific) cytotoxicity        against these cancer-specific antigen-expressing cells or tumor        tissues containing these cells. A cancer-specific        antigen-binding domain, TNF superfamily-binding domain, and TNF        receptor superfamily-binding domain of the present invention can        be appropriately selected for the above-described        cancer-specific antigens or antigens belonging to the TNF        superfamily or the TNF receptor superfamily, respectively. These        binding domains can be linked directly by peptide bonds or        connected via linkers.

In several embodiments, the first immunoactivating antigen-bindingmolecules of the present invention may further comprise an FcRn-bindingdomain. When using an antibody Fc region described above as theFcRn-binding domain, Fc regions having decreased Fcγ receptor-bindingactivity are preferred. Reducing the Fcγ receptor-binding activityenables suppression of side effects produced by immune activation suchas cytokine release caused by crosslinking between Fcγreceptor-expressing cells and cells that express factors belonging tothe TNF superfamily or the TNF receptor superfamily.

The first immunoactivating antigen-binding molecules of the presentinvention can be produced using known methods described above. Forexample, when (1) using F(ab′)2 as the cancer-specific antigen-bindingdomain, (2) using F(ab′)2 as the TNF superfamily-binding domain or theTNF receptor superfamily-binding domain, and (3) using a domaincomprising an Fc region with decreased Fcγ receptor-binding activity asthe FcRn-binding domain, and when the antigen-binding domains describedin (1) and (2) and the Fc region-containing domain described in (3) aredirectly linked by peptide bonds, the linked polypeptides will form anantibody structure. To produce such antibodies, antibodies can bepurified from the afore-mentioned hybridoma culture medium, and alsopurified from the culture medium of desired host cells that stably carrypolynucleotides encoding polypeptides constituting the antibodies.

In addition to the linkers exemplified above, linkers with peptide tagssuch as His tag, HA tag, myc tag, and FLAG tag may also be suitably usedas the linkers to be employed when connecting each of the domains vialinkers. Furthermore, the property of mutual binding by hydrogenbonding, disulfide bonding, covalent bonding, ionic interaction, orcombination thereof may be suitably used. For example, the affinitybetween antibody CH1 and CL may be used, and Fc regions derived from theabove-described multispecific antibodies may also be used forheterologous Fc region association.

In several embodiments, for example, the “first antigen-bindingmolecules” disclosed in WO2015/156268 may be used favorably as the firstimmunoactivating antigen-binding molecules described above.

In several embodiments, an example of anticancer agents of the presentinvention includes an immunoactivating antigen-binding moleculedifferent from that described above (a second immunoactivatingantigen-binding molecule). In several embodiments, as in the case withthe first immunoactivating antigen-binding molecules, the structure ofthe second immunoactivating antigen-binding molecules is not limited andthey may be any molecule as long as they comprise:

-   -   (1) a cancer-specific antigen-binding domain, and    -   (2) a T cell receptor complex-binding domain;        and they can be obtained by methods similar to those for the        first immunoactivating antigen-binding molecules. Furthermore,        as long as the second immunoactivating antigen-binding molecules        contain a cancer-specific antigen-binding domain and a T cell        receptor complex-binding domain, their structure does not have        to be the same as that of the first immunoactivating        antigen-binding molecules. A cancer-specific antigen bound by        the cancer-specific antigen-binding domain of the first        immunoactivating antigen-binding molecules may be the same as or        different from a cancer-specific antigen bound by the        cancer-specific antigen-binding domain of the second        immunoactivating antigen-binding molecules.

Similarly to the first antigen-binding molecules, the secondantigen-binding molecules of the present invention may further comprisean FcRn-binding domain. When an antibody Fc region described above isused as the FcRn-binding domain, Fc regions having decreased Fcγreceptor-binding activity are preferred, as in the case of the firstantigen-binding molecules. Reducing the Fcγ receptor-binding activityenables suppression of side effects produced by immune activation suchas cytokine release caused by crosslinking between Fcγreceptor-expressing cells and T cell receptor complex-expressing cells.

In several embodiments, for example, the “second antigen-bindingmolecules” disclosed in WO2015/156268 may be used favorably as thesecond immunoactivating antigen-binding molecules described above.

Furthermore, in other embodiments, the second immunoactivatingantigen-binding molecules may be a molecule that comprises:

-   -   (1) an antigen-binding domain towards an antigen other than a        cancer-specific antigen; and    -   (2) a T cell receptor complex-binding domain.        For example, the “polypeptide complexes” disclosed in        WO2012/073985 may be used favorably as the second        immunoactivating antigen-binding molecules comprising the        “antigen-binding domain towards an antigen other than a        cancer-specific antigen” mentioned above.

In several embodiments, an antigen of the above-mentioned“antigen-binding domain towards an antigen other than a cancer-specificantigen” is not particularly limited, and may be any antigen excludingCD3. Examples of the antigens favorably include receptors, MHC antigens,and differentiation antigens. For example, the receptors, MHC antigens,differentiation antigens, and such disclosed in WO2012/073985 may befavorably used.

Combination Therapies

In several embodiments, a combination therapy of the present inventionprovides methods for damaging cells, for suppressing cell proliferation,for activating immunity towards cancer cells or cancer cell-comprisingtumor tissues, for treating cancer, or for preventing cancer, each ofthe methods comprising administering effective amounts of an anticanceragent and a T cell-redirecting antigen-binding molecule. In severalembodiments, a combination therapy of the present invention is highlyeffective for damaging cells, suppressing cell proliferation, activatingimmunity towards cancer cells or cancer cell-comprising tumor tissues,treating cancer, or preventing cancer, as compared to monotherapy usingan anticancer agent or a T cell-redirecting antigen-binding molecule. Inanother embodiment, a combination therapy of the present invention hassynergistic effects or additive effects on damaging cells, suppressingcell proliferation, activating immunity towards cancer cells or cancercell-comprising tumor tissues, treating cancer, or preventing cancer.

In several embodiments, the term “effective amount” according to thepresent invention refers to a dose of a T cell-redirectingantigen-binding molecule and/or an anticancer agent effective fortreating or preventing a disease in individuals. The disease is notparticularly limited but is preferably cancer.

In several embodiments, “treatment/treating/therapeutic” according tothe present invention means that a combination therapy of the presentinvention decreases the number of cancer cells in individuals,suppresses cancer cell proliferation, decreases tumor size, suppressesinfiltration of cancer cells into peripheral organs, suppresses cancercell metastasis, or ameliorates various symptoms caused by cancer.Furthermore, in several embodiments,“prevention/preventing/prophylactic” according to the present inventionrefers to inhibiting increase in the number of cancer cells due torepopulation of cancer cells that have decreased, inhibitingrepopulation of cancer cells whose proliferation have been suppressed,and inhibiting the decreased tumor size to become large again.

In several embodiments, a combination therapy of the present inventionprovides a method for enhancing therapeutic or prophylactic effects ofan anticancer agent by using a T cell-redirecting antigen-bindingmolecule in cancer treatment or prevention with the anticancer agent. Inanother embodiment, a combination therapy of the present inventionprovides a method for enhancing therapeutic or prophylactic effects of aT cell-redirecting antigen-binding molecule by using an anticancer agentin cancer treatment with the T cell-redirecting antigen-bindingmolecule. Herein, enhancement of therapeutic or prophylactic effectsrefers to, for example, increase in efficacy rate of the treatment,decrease in the amount of an anticancer agent that is administered forthe treatment, and/or shortening of the period of the treatment with ananticancer agent, but is not limited thereto. In another embodiment, acombination therapy of the present invention provides a method forextending the progression-free survival in individuals, the methodcomprising administering an effective amount of a T cell-redirectingantigen-binding molecule and an anticancer agent.

In several embodiments, an “individual” to which an antigen-bindingmolecule and an anticancer agent are administered refers to a human or anon-human mammal such as a mammal including cattle, horse, dog, sheep,or cat. Preferably, the individuals are humans. Individuals includepatients (including humans and non-human mammals). In severalembodiments, the individuals are patients who have cancer cells orcancer cell-comprising tumor tissues. Such tumor cells are notparticularly limited, but they are preferably cancer types in whichcells having immune response-suppressing activity are involved in cancerprogression, or cancer types in which the number of regulatory T cellsor exhausted T cells in the tumor correlates with the prognosis.Reported examples of such cancer types include ovarian cancer(Non-patent Documents 15 and 16), gastric cancer (Non-patent Documents17 and 18), esophageal cancer (Non-patent Document 18), pancreaticcancer (Non-patent Document 19), renal cell carcinoma (Non-patentDocument 20), hepatocellular carcinoma (Non-patent Document 21), breastcancer (Non-patent Documents 22 and 23), malignant melanoma (Non-patentDocument 24), non-small-cell lung cancer (Non-patent Document 25),cervical cancer (Non-patent Document 26), glioblastoma (Non-patentDocument 27), prostate cancer (Non-patent Document 28), neuroblastoma(Non-patent Document 29), chronic lymphocytic leukemia (Non-patentDocument 30), papillary thyroid cancer (Non-patent Document 31),colorectal cancer (Non-patent Document 32), and B-cell non-Hodgkin'slymphoma (Non-patent Documents 33 and 34), and they are suitableexamples of cancer types for the present invention. In severalembodiments, the patients are those who have been treated with a Tcell-redirecting antigen-binding molecule and/or some kind of ananticancer agent prior to combination therapy using a T cell-redirectingantigen-binding molecule and an anticancer agent. In severalembodiments, the patients have early-stage or end-stage cancer.

In several embodiments, a combination therapy of the present inventioncomprises administering a T cell-redirecting antigen-binding moleculeand an anticancer agent. The T cell-redirecting antigen-binding moleculeand the anticancer agent can be administered by any appropriate methodknown in the art. For example, the T cell-redirecting antigen-bindingmolecule and the anticancer agent can be administered in parallel (i.e.,simultaneously), or successively (i.e., at different time points). Inseveral embodiments, when administering the T cell-redirectingantigen-binding molecule and the anticancer agent successively (i.e., atdifferent time points), the interval between administration of the Tcell-redirecting antigen-binding molecule and the anticancer agent isnot particularly limited, and the interval can be determined by takingaccount of factors such as the administration route and dosage form. Forexample, the interval is 0 to 168 hours, preferably 0 to 72 hours, morepreferably 0 to 24 hours, and even more preferably 0 to 12 hours, but isnot limited thereto.

In several embodiments, the T cell-redirecting antigen-binding moleculeand the anticancer agent are administered simultaneously. In severalembodiments, the T cell-redirecting antigen-binding molecule isadministered at intervals (i.e., intermittently). In some embodiments,the T cell-redirecting antigen-binding molecule is administered beforeadministration of the anticancer agent. In several embodiments, the Tcell-redirecting antigen-binding molecule is administered afteradministration of the anticancer agent.

In several embodiments, the anticancer agent is administered atintervals (i.e., intermittently). In some embodiments, the anticanceragent is administered before administration of the T cell-redirectingantigen-binding molecule. In several embodiments, the anticancer agentis administered after administration of the T cell-redirectingantigen-binding molecule.

In several embodiments, the T cell-redirecting antigen-binding moleculeand the anticancer agent can be administered through the sameadministration route or different administration routes. In severalembodiments, the T cell-redirecting antigen-binding molecule and/or theanticancer agent can be administered to patients by either oral orparenteral administration. Specific examples of methods for parenteraladministration include administration by injection, transnasaladministration, transpulmonary administration, and transdermaladministration. Examples of administration by injection includeintravenous injection, intramuscular injection, intraperitonealinjection, and subcutaneous injection. The T cell-redirectingantigen-binding molecule and/or the anticancer agent can be administeredsystemically or locally, for example, through administration byinjection. The dose can be selected, for example, from the range of0.0001 mg to 1000 mg per kilogram body weight for a singleadministration. Alternatively, for example, the dose may be selectedfrom the range of 0.001 mg/body to 100000 mg/body per patient. However,the method of administration and dose of the T cell-redirectingantigen-binding molecule and/or the anticancer agent in combinationtherapy are not limited to the above, and a suitable method foradministration and dose can be selected according to the patient's ageand symptoms. Furthermore, when the T cell-redirecting antigen-bindingmolecule and the anticancer agent are used in combination according tothe present invention, the respective doses may be reduced, if desired,as compared to when either one of them is used alone.

In several embodiments, T cell-redirecting antigen-binding moleculesdescribed herein and anticancer agents which are known or describedherein may be used in the above-mentioned combination therapy using a Tcell-redirecting antigen-binding molecule and an anticancer agent.

Additional Combination Therapies

In several embodiments, in addition to the combination therapy using a Tcell-redirecting antigen-binding molecule and an anticancer agent,additional therapy can be carried out. In several embodiments, a therapyto be added to the combination therapy of the present invention maycomprise additional administration of a T cell-redirectingantigen-binding molecule and/or an anticancer agent. For example, inaddition to a combination therapy using a T cell-redirectingantigen-binding molecule and a first immunoactivating antigen-bindingmolecule, a second immunoactivating antigen-binding molecule may besuitably used.

Pharmaceutical Compositions

In several embodiments, the present invention provides agents forinducing cytotoxicity, agents for suppressing cell proliferation (agentsfor inhibiting cell proliferation), agents for activating immuneresponse towards cancer cells or cancer cell-comprising tumor tissues,and agents for treating or preventing cancer (hereinafter,pharmaceutical compositions, and such), each comprising a Tcell-redirecting antigen-binding molecule, an anticancer agent, or acombination of a T cell-redirecting antigen-binding molecule and ananticancer agent. In several embodiments, pharmaceutical compositionsand such of the present invention may be used in a combination therapyof the present invention. In several embodiments, pharmaceuticalcompositions and such of the present invention are highly effective fordamaging cells, suppressing cell proliferation, activating immunitytowards cancer cells or cancer cell-comprising tumor tissues, treatingcancer, or preventing cancer through combined use of a Tcell-redirecting antigen-binding molecule and an anticancer agent, ascompared to monotherapy using the T cell-redirecting antigen-bindingmolecule or the anticancer agent. In another embodiment, pharmaceuticalcompositions of the present invention have synergistic effects oradditive effects on damaging cells, suppressing cell proliferation,activating immunity towards cancer cells or cancer cell-comprising tumortissues, treating cancer, or preventing cancer through combined use of aT cell-redirecting antigen-binding molecule and an anticancer agent.

In several embodiments, pharmaceutical compositions and such accordingto the present invention “comprising a combination of a Tcell-redirecting antigen-binding molecule and an anticancer agent” referto pharmaceutical compositions and such in which the T cell-redirectingantigen-binding molecule and the anticancer agent are combined for usein simultaneous, separate, or sequential administration in treatment orprevention of a disease. For example, pharmaceutical compositions andsuch of the present invention can be provided in the form of acombination preparation comprising both a T cell-redirectingantigen-binding molecule and an anticancer agent. Alternatively, forexample, as the pharmaceutical compositions and such of the presentinvention a pharmaceutical agent containing a T cell-redirectingantigen-binding molecule and a pharmaceutical agent containing ananticancer agent can be separately provided, and these pharmaceuticalagents can be used simultaneously or sequentially. The disease mentionedabove is not particularly limited, but is preferably cancer.

In several embodiments, the present invention provides pharmaceuticalcompositions and such for use in combination with an anticancer agent,the compositions comprising a T cell-redirecting antigen-bindingmolecule as an active ingredient.

In several embodiments, the present invention provides pharmaceuticalcompositions and such for use in combination with a T cell-redirectingantigen-binding molecule, the compositions comprising an anticanceragent as an active ingredient.

In several embodiments, the present invention provides pharmaceuticalcompositions and such for enhancing therapeutic effects of an anticanceragent in cancer treatment with the anticancer agent, by using a Tcell-redirecting antigen-binding molecule in combination with theanticancer agent.

In several embodiments, the present invention provides pharmaceuticalcompositions and such for enhancing therapeutic effects of a Tcell-redirecting antigen-binding molecule in cancer treatment with the Tcell-redirecting antigen-binding molecule, by using an anticancer agentin combination with the T cell-redirecting antigen-binding molecule.

In several embodiments, the present invention provides use of a Tcell-redirecting antigen-binding molecule and/or an anticancer agent forthe production of pharmaceutical compositions and such comprising asactive ingredients a T cell-redirecting antigen-binding molecule and/oran anticancer agent.

In the present invention, “comprising as active ingredients a Tcell-redirecting antigen-binding molecule and/or an anticancer agent”means containing a T cell-redirecting antigen-binding molecule and/or ananticancer agent as major active component(s), and does not limit thecontent of the T cell-redirecting antigen-binding molecule and/or theanticancer agent.

In several embodiments, T cell-redirecting antigen-binding moleculesdescribed herein and anticancer agents which are known or describedherein may be used in the pharmaceutical compositions and such describedabove.

Methods for Contacting a T Cell-Redirecting Antigen-Binding Molecule andan Anticancer Agent with Cancer Cells.

In several embodiments, the present invention provides methods forinducing damage to cancer cells or cancer cell-comprising tumor tissues,or methods for suppressing proliferation of cancer cells or cancercell-comprising tumor tissues, by contacting the cancer cells and thecells having an immune response-suppressing function with anticanceragents and T cell-redirecting antigen-binding molecules that bind tomolecules expressed on the surface of the cells having an immuneresponse-suppressing function. In another embodiment, the presentinvention provides methods for assessing whether T cell-redirectingantigen-binding molecules and anticancer agents induce damage to cancercells or cancer cell-comprising tumor tissues or suppress proliferationof cancer cells or cancer cell-comprising tumor tissues, by contactingthe cancer cells and the cells having an immune response-suppressingfunction with the anticancer agents and the T cell-redirectingantigen-binding molecules that bind to molecules expressed on thesurface of the cells having an immune response-suppressing function. Thecancer cells to be contacted with T cell-redirecting antigen-bindingmolecules and anticancer agents are not particularly limited, but forexample, cancer types which become targets of combination therapy of thepresent invention may be used favorably.

In several embodiments, “contact” according to the present invention iscarried out, for example, by adding an anticancer agent and a Tcell-redirecting antigen-binding molecule that binds to an antigen to aculture medium of in vitro cultured cancer cells or cancercell-comprising tumor tissues, and/or cells expressing an antigen whichis the binding target of the T cell-redirecting antigen-bindingmolecules. In this case, the T cell-redirecting antigen-binding moleculeand the anticancer agent to be added may be mixed, or they may beseparate. Furthermore, the T cell-redirecting antigen-binding moleculesor the anticancer agents to be added may be individually used in asuitable form such as a solution, or a solid obtained by freeze-dryingand the like. When added as an aqueous solution, an aqueous solutioncontaining purely the T cell-redirecting antigen-binding molecule or theanticancer agent alone may be used, or a solution containingsurfactants, excipients, coloring agents, perfumes, preservatives,stabilizers, buffers, suspending agents, isotonization agents, binders,disintegrants, lubricants, fluidity promoting agents, flavoring agents,and such described above may be used. The concentration of the Tcell-redirecting antigen-binding molecule or the anticancer agent to beadded is not particularly limited, but a suitable final concentration inthe culture solution may be preferably in the range of 1 pg/ml to 1g/ml, more preferably 1 ng/ml to 1 mg/ml, and even more preferably 1μg/ml to 1 mg/ml, individually.

In several embodiments, “contact” according to the present invention iscarried out, for example, by administering to a non-human animal intowhich a cancer cell line of interest has been transplanted, ananticancer agent and a T cell-redirecting antigen-binding molecule thatbinds to a molecule expressed on the surface of a cell having thefunction of suppressing the immune response in the non-human animal; orby transplanting a cancer cell line of interest into a non-human animalexpressing human-derived cells having an immune response-suppressingfunction, and then administering to the animal an anticancer agent and aT cell-redirecting antigen-binding molecule that binds to a moleculeexpressed on the surface of the human-derived cells. The method ofadministration and the dose are not particularly limited, butadministration methods and doses used in combination therapies of thepresent invention may be suitably used.

In several embodiments, examples of in vitro methods for evaluating ormeasuring the effects of damaging cancer cells or cancer cell-comprisingtumor tissues, or the effects of suppressing proliferation of cancercells or cancer cell-comprising tumor tissues include methods formeasuring cytotoxic T cell activity, and such. Whether anantigen-binding molecule of the present invention has cytotoxic T cellactivity can be measured by known methods (for example, Currentprotocols in Immunology, Chapter 7. Immunologic studies in humans,Editor, John E. Coligan et al., John Wiley & Sons, Inc., (1993)). As aresult of the measurements, for example, when the cytotoxic T cellactivity in the group of combined use of a T cell-redirectingantigen-binding molecule and an anticancer agent is stronger than thecytotoxic T cell activity in the control group, the group to which the Tcell-redirecting antigen-binding molecule is administered alone, or thegroup to which the antitumor agent is administered alone, combinedadministration of the T cell-redirecting antigen-binding molecule andthe anticancer agent can be determined as being highly effective for orhaving synergistic effects or additive effects on damaging cancer cellsor cancer cell-comprising tumor tissues or suppressing proliferation ofcancer cells or cancer cell-comprising tumor tissues.

In several embodiments, for in vivo evaluation or measurement of theeffects of damaging cancer cells or cancer cell-comprising tumortissues, or the effects of suppressing proliferation of cancer cells orcancer cell-comprising tumor tissues, for example, cancer cells orcancer cell-comprising tumor tissues are intradermally or subcutaneouslytransplanted to a non-human test animal, and then a T cell-redirectingantigen-binding molecule and/or an anticancer agent are intravenously orintraperitoneally administered daily or at intervals of a few days,starting from the day of transplantation or the following day. Tumorsize is measured daily and the differences in the change of tumor sizecan be defined as the effects of damaging cancer cells or cancercell-comprising tumor tissues, or the effects of suppressingproliferation of cancer cells or cancer cell-comprising tumor tissues.For determination of the above-mentioned effects, for example, a controlantigen-binding molecule, a T cell-redirecting antigen-binding moleculeand/or an anticancer agent are administered, and the tumor size in eachof the administered groups can be measured. As a result of themeasurements, for example, when the tumor size in the group of combineduse of a T cell-redirecting antigen-binding molecule and an anticanceragent is smaller than the tumor size in the control group, the group towhich the T cell-redirecting antigen-binding molecule is administeredalone, or the group to which the anticancer agent is administered alone,the combined administration of the T cell-redirecting antigen-bindingmolecule and the anticancer agent can be determined as being highlyeffective for or having synergistic effects or additive effects ondamaging cancer cells or cancer cell-comprising tumor tissues orsuppressing proliferation of cancer cells or cancer cell-comprisingtumor tissues. Kits

In several embodiments, the present invention provides a kit comprising(1) a T cell-redirecting antigen-binding molecule, (2) a container, and(3) an instruction or a label indicating that the T cell-redirectingantigen-binding molecule and at least one type of anticancer agent areadministered in combination to a subject individual for treating cancerin the individual. In another embodiment, the present invention providesa kit comprising (1) an anticancer agent, (2) a container, and (3) aninstruction or a label indicating that the anticancer agent and at leastone type of T cell-redirecting antigen-binding molecule are administeredin combination to an individual for treating cancer in the individual.

In another embodiment, the present invention provides a kit comprising(1) a T cell-redirecting antigen-binding molecule, (2) an anticanceragent, (3) a container, and (4) an instruction or a label indicatingthat the T cell-redirecting antigen-binding molecule and the anticanceragent are administered in combination to an individual for treatingcancer in the individual.

In several embodiments, the kit further comprises a pharmaceuticallyacceptable carrier. Additionally, the kit may further comprise asterilized diluent preferably stored in a separate additional container.The kit may also comprise an instruction relating to combination therapyfor treating or preventing cancer.

In several embodiments, an “instruction” refers to the writteninstruction usually contained in the commercially available box carryinga pharmaceutical, and may contain information on indications, usage,dosage, administration, contraindications and/or warnings regarding theuse of the pharmaceutical.

Those skilled in the art will naturally understand that optionalcombinations of one or more of the embodiments described herein areincluded in the present invention, as long as they are not technicallyinconsistent based on common technical knowledge of those skilled in theart.

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLES [Reference Example 1] Concept of T Cell-Redirecting AntibodyTargeting a Cell Surface Marker of a Regulatory T Cell (1-1) AntitumorEffects of Anti-CTLA4 Antibodies Through Elimination of Regulatory TCells

As described above in Background Art, Ipilimumab had been considered toinhibit CTLA4 expressed on the surface of effector T cells fromsuppressing effector T-cell activation, and thereby exhibit antitumoreffects However, recently, antibody-dependent cellular cytotoxicactivity (ADCC activity) against CTLA4-expressing T cells was alsoreported to be important, and elimination of regulatory T cells intumors and ADCC activity have been found to be important mechanisms ofaction for the antitumor effects of anti-CTLA4 antibodies.

On the other hand, ADCC activity by an IgG1 antibody induces cytotoxicactivity through binding of the antibody constant region to FcγR of NKcells or macrophages, and antibodies having a constant region that havebeen modified so as to enhance such binding are known to induce strongercytotoxic activities and demonstrate antitumor effects.

As mentioned above, binding to regulatory T cells or exhausted T cellsin a cancer microenvironment, and elimination of regulatory T cells orexhausted T cells by cytotoxic activity were found to exert strongantitumor effects. Therefore, if regulatory T cells or exhausted T cellscan be eliminated more powerfully, or more specifically, if strongercytotoxic activity can be exhibited, stronger antitumor effects can beexpected to be exerted.

(1-2) T Cell-Redirecting Antibody

Enhancement of the aforementioned ADCC activity, increase of retentionin blood, improvement of antigen-binding activity, and reduction ofimmunogenicity risk have been performed as techniques for improvingantibodies. Generally, antibodies recognize and bind a single epitope ofan antigen, therefore even when such techniques for improvement areapplied to the antibodies, only one type of antigen becomes the target.As molecules that inhibit multiple targets, antibodies that bind to twoor more types of antigens by one molecule (referred to as bispecificantibodies) have been studied. Since bispecific antibodies interact withtwo or more types of antigens, they have not only the effect ofneutralizing two or more types of antigens with one molecule, but alsothe effect of enhancing antitumor activities by crosslinking cellshaving cytotoxic activity with cancer cells.

Blinatumomab, which is a BiTE molecule, and Catumaxomab are known asbispecific antibodies that recognize a protein expressed on T cells(CD3e or TCR) and a protein expressed on cancer cells (a cancerantigen). These molecules can bind to a cancer antigen and the CD3echain expressed on a T cell with each of their two antigen-bindingdomains (scFv or Fab), and form intercellular crosslinks between the Tcells and the cancer antigen-expressing cells (FIG. 1). This way, such Tcell-redirecting antibodies can use T cells as effector cells to inducestrong cytotoxic activity against cancer antigen-expressing cells.

Meanwhile, antibodies that use T cells as effector cells and inducestrong cytotoxic activity against T cells have not been reported so far.For example, since CD3ε is a standard T cell marker, an IgG antibody(not having FcgR-binding activity) which binds to CD3εs using both arms(two Fabs) may be able to cause T cells to induce strong cytotoxicactivity against T cells by forming intercellular crosslink between aCD3ε-expressing T cell which will become an effector cell and aCD3ε-expressing T cell, as shown in FIG. 2-1, but this hardly occurs inreality. That is because an IgG antibody that binds to CD3εs using botharms (two Fabs) strongly binds to CD3εs expressed on the same cell dueto avidity via bivalent binding, intercellular crosslink is not formedbetween CD3ε-expressing T cells (FIG. 2-2).

Since CD3 is a standard T cell marker and it is expressed in both Tcells which will become the effector cells and T cells which will becomethe target (for example, regulatory T cells and exhausted T cells), Tcell-redirecting antibodies had been considered to be not able to exertcytotoxic activity against target T cells. More specifically, a Tcell-redirecting antibody against a T cell expressing antigen X, asurface marker of a specific T cell population (bispecific antibodyagainst CD3ε and antigen X) strongly binds to the target T cell (forexample, a regulatory T cell and an exhausted T cell) with avidity viabivalent binding, since CD3ε and antigen X are expressed on the target Tcell. Therefore, intercellular crosslinking was considered not to takeplace with T cells which will become effector cells (FIG. 3). In fact,there have been no reports of T cell-redirecting antibodies against Tcell antigens.

Therefore, T cell-redirecting antibodies against CTLA4 expressed onregulatory T cells and exhausted T cells (bispecific antibodies againstCD3ε and CTLA4), as shown in FIG. 4, are considered not to be able toinduce strong cytotoxic activity against regulatory T cells andexhausted T cells because both CD3ε and CTLA4 are expressed onregulatory T cells and exhausted T cells, which are target cells, andthe T cells which will become effector cells are not crosslinked withregulatory T cells and exhausted T cells. So far, there have been noreports on T cell-redirecting antibodies against regulatory T cells orexhausted T cells.

More specifically, it was unknown whether T cell-redirecting antibodiesagainst CTLA4 (anti-CTLA4/anti-CD3ε bispecific antibodies) can actuallydamage regulatory T cells by inducing intercellular crosslinking anddemonstrate antitumor effects in vivo. Therefore, we actually producedbispecific antibodies against CTLA4 and CD3 and tested whether they canachieve effects in vivo in mice and in vitro in humans.

Against standard cancer antigens, T cell-redirecting antibodies havebeen known to have stronger antitumor effects than antibodies having NKcell-utilizing ADCC activity; however, it had been unknown whether Tcell-redirecting antibodies against CTLA4 show stronger antitumoreffects than anti-CTLA4 antibodies with enhanced ADCC activity.

[Reference Example 2] Preparation and Assessment of ADCCActivity-Enhanced Antibodies Targeting Regulatory T Cells

(2-1) Expression and Purification of an ADCC Activity-Enhanced Antibodythat Binds Specifically to Mouse CTLA4 (hUH02hUL01-mFa55)

Genes encoding the variable regions of the anti-mouse CTLA4 antibodyhUH02hUL01 (the heavy chain variable region UH02 is SEQ ID NO: 28, andthe light chain variable region UL01 is SEQ ID NO: 29) were eachinserted into mouse IgG2a/kappa plasmids for expression in animals.Here, constant regions that have been modified so as to enhance bindingto mouse FcγR were used (the heavy chain constant region mFa55 is SEQ IDNO: 30, and the light chain constant region mk1 is SEQ ID NO: 31).

Antibodies were expressed by the method described below. Cells of humanembryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) weresuspended in FreeStyle 293 Expression Medium (Invitrogen) at a celldensity of 1.33×10⁶ cells/mL, and seeded into each well of a 6-wellplate at 3 mL/well. The prepared plasmids were introduced into the cellsby a lipofection method. The cells were cultured for four days in a CO₂incubator (37° C., 8% CO₂, 90 rpm). From the culture supernatants,antibodies were purified using rProtein A Sepharose™ Fast Flow (AmershamBiosciences) by a method known to those skilled in the art. Absorbanceat 280 nm of the purified antibody solutions was measured using aspectrophotometer. Concentrations of the purified antibodies werecalculated from the determined values using an extinction coefficientcalculated by the PACE method (Protein Science (1995) 4: 2411-2423).

(2-2) Assessment of Binding of the Anti-Mouse CTLA4 Antibody(hUH02UL01-mFa55) to Various Mouse FcgRs

Anti-mouse CTLA4 antibody hUH02hUL01-mFa55 and control antibodyhUH02hUL01-mIgG2a (the heavy chain variable region UH02 is SEQ ID NO:28, the light chain variable region UL01 is SEQ ID NO: 29, the heavychain constant region mIgG2a is SEQ ID NO: 32, and the light chainconstant region mk1 is SEQ ID NO: 31) purified and prepared by themethod of Reference Example 2-1 were analyzed for their antigen-antibodyreactions with various mouse FcgRs (mFcgRI, II, III, and IV) usingBiacore T200 (GE Healthcare). The running buffer used was 20 mmol/LACES, 150 mmol/L NaCl, 0.05% (w/v) Tween20 at pH7.4, and measurementswere taken at 25° C. Protein A/G was immobilized onto a Sensor Chip CM7by amine coupling. hUH02hUL01-mFa55 was captured onto the sensorchip,and then FcgR was allowed to interact as an analyte for 120 seconds, andchange in the bound amount was observed. The running buffer was used fordilution of hUH02hUL01-mFa55. The measurement results were analyzed bycurve fitting using the Biacore T200 Evaluation Software (GE Healthcare)to calculate the association rate constants ka (1/Ms) and thedissociation rate constants kd (l/s). From those values, thedissociation constants K_(D) (M) were determined. The measurementresults are shown in Table 1.

TABLE 1 mFcgRI mFcgRII 1:1 binding RI = 0 1:1 binding RI = 0 Name ka kdKD ka kd KD No. of Fc (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 1 mIgG2a 7.8E+054.7E−03 6.1E−09 1.3E+06 1.1E+00 7.8E−07 2 mFa55 1.2E+06 3.1E−03 2.5E−092.1E+06 7.6E−01 3.7E−07 mFcgRIII mFcgRIV 1.1 binding RI = 0 1:1 bindingRI = 0 Name ka kd KD ka kd KD No. of Fc (1/Ms) (1/s) (M) (1/Ms) (1/s)(M) 1 mIgG2a 2.0E+06 5.1E−01 2.5E−07 1.2E+06 1.6E−02 1.3E−08 2 mFa552.4E+06 6.2E−01 2.6E−07 1.5E+06 2.8E−03 1.9E−09(2-3) Evaluation of an Anti-Mouse CTLA4 Antibody (hUH02UL01-mFa55) as anADCC Activity-Enhanced Antibody

Whether anti-mouse CTLA4 antibody hUH02hUL01-mFa55 purified and preparedby the method of Reference Example 2-1 exerts ADCC activity on mouseCTLA4-expressing cells (mouse CTLA4-expressing cells were produced by amethod known to those skilled in the art by introducing the full lengthmouse CTLA4 gene into CHO cells) was examined according to the method ofReference Example 11. The measurement results show ADCC activity in anantibody concentration-dependent manner (FIG. 5).

[Reference Example 3] Preparation and Assessment of Bispecificity whichRecognizes Surface Antigens of Regulatory T Cells and Effector T Cells

(3-1) Expression and Purification of Bispecific Antibodies thatSpecifically Bind to Mouse CTLA4 and Mouse CD3

Genes encoding the variable regions of the anti-mouse CTLA4 antibodyhUH02hUL01 (the heavy chain variable region UH02 is SEQ ID NO: 28, andthe light chain variable region UL01 is SEQ ID NO: 29) were eachinserted into human IgG1/kappa plasmids for expression in animals. Here,constant regions that have been modified so as to reduce binding to Fcγreceptors and to produce heterologous association of two heavy chainswere used (the heavy chain constant region F760nN17 is SEQ ID NO: 33,and the light chain constant region k0 is SEQ ID NO: 34).

Genes encoding the variable regions of the anti-mouse CD3 antibody 2C11(the heavy chain variable region is SEQ ID NO: 35, and the light chainvariable region is SEQ ID NO: 36) were each inserted into humanIgG1/kappa plasmids for expression in animals. Here, constant regionsthat have been modified so as to reduce binding to Fcγ receptors and toproduce heterologous association of two heavy chains were used (theheavy chain constant region F760nP17 is SEQ ID NO: 37, and the lightchain constant region k0 is SEQ ID NO: 34).

Each of hUH02hUL01-F760nN17 and 2C11-F760nP17 was expressed and purifiedby the method shown in Reference Example 2. Each of the purifiedhomologous forms was mixed by a method known to those skilled in the artthat uses differences in the charges of the constant regions (Proc.Natl. Acad. Sci., 110, 5145-5150, 2013) to produce the bispecificantibody of interest (hUH02UL01/2C11-F760).

(3-2) Evaluation of a Bispecific Antibody that Specifically Binds toMouse CTLA4 and Mouse CD3 for the Antigen (mCTLA4 and mCD3)-BindingProperties

Anti-mouse CTLA4/anti-mouse CD3 bispecific antibodies(hUH02UL01/2C11-F760) purified and prepared by the method of ReferenceExample 3-1 were analyzed for their antigen-antibody reactions with eachantigen (mCTLA4 and mCD3) using Biacore T200 (GE Healthcare). Therunning buffer used was HBS-EP+ at pH7.4, and measurements were taken at37° C. Protein A/G was immobilized onto a Sensor Chip CM4 by aminecoupling. hUH02UL01/2C11-F760 was captured onto the sensor chip, andthen the antigen (mouse CTLA4 or mouse CD3) was allowed to interact asan analyte (for 120 seconds for mouse CTLA4 and for 90 seconds for mouseCD3), and changes in the bound amount were observed. The running bufferwas used for dilution of hUH02UL01/2C11-F760. The measurement resultswere analyzed by curve fitting using the Biacore T200 EvaluationSoftware (GE Healthcare) to calculate the association rate constants ka(1/Ms) and the dissociation rate constants kd (l/s). From those values,the dissociation constants KD (M) were determined. The results are shownin Table 2.

TABLE 2 No Analyte ka (1/Ms) kd (1/s) KD (M) 1 MouseCTLA4 4.30E+055.30E−04 1.23E−09 2 Mouse CD3 6.16E+04 7.12E−02 1.16E−06(3-2) Evaluation of Cytotoxic Activity by an Anti-Mouse CTLA4/Anti-MouseCD3 Bispecific Antibody (hUH02UL01/2C11-F760)

Whether anti-mouse CTLA4/anti-mouse CD3 bispecific antibody(hUH02UL01/2C11-F760) purified and prepared by the method of ReferenceExample 3-1 exerts cytotoxic activity on mouse CTLA4-expressing celllines was examined according to the method of Reference Example 12. Themeasurement results show cytotoxic activity in an antibodyconcentration-dependent manner (FIG. 6).

[Reference Example 4] Assessment of Crosslinking Between Beads to whichCD3 and CTLA4 have been Immobilized and CD3-Bound Beads by an Anti-MouseCTLA4/Anti-Mouse CD3 Bispecific Antibody (hUH02UL01/2C11-F760)

Whether an anti-CTLA4/anti-CD3 bispecific antibody recognizes surfaceantigens of a regulatory T cell (expressing CTLA4 and CD3) and aneffector T cell (expressing CD3) and forms a crosslink between the twocells was verified by a physicochemical experiment.

First, the present inventors investigated, using Alpha technology fromPerkin Elmer Inc., construction of a system that can evaluatecrosslinking between mouse CD3-immobilized beads and mouseCTLA4-immobilized beads using the anti-mouse CTLA4/anti-mouse CD3bispecific antibody (hUH02UL01/2C11-F760) purified and prepared by themethod of Reference Example 3-1. More specifically, 100 nmol/L ofbiotinylated mouse CTLA4; 0, 20, 100, or 500 nmol/L ofhUH02UL01/2C11-F760; 50 μg/mL of AlphaScreen (registered trademark)Streptavidin-coated Donor Beads (PerkinElmer); and mouse CD3-acceptorbeads prepared by conjugating 50 μg/mL of mouse CD3 to AlphaScreen(registered trademark) Unconjugated Acceptor Beads (PerkinElmer) wereused. Under this condition, it was thought that biotinylated mouse CTLA4bound to AlphaScreen (registered trademark) Streptavidin-coated DonorBeads and mouse CD3-acceptor beads may be crosslinked byhUH02UL01/2C11-F760 as shown in FIG. 7-1, and chemiluminescence may beemitted. Alpha 384 (PerkinElmer) was used as the plate, and themeasurements were taken using Envision. All experiments were performedthree times. As a result, hUH02UL01/2C11-F760 concentration-dependentcrosslinking between beads was observed as shown in FIG. 7-2.

Next, 100 nmol/L of biotinylated mouse CTLA4 and 10 nmol/L ofbiotinylated mouse CD3 were added to AlphaScreen (registered trademark)Streptavidin-coated Donor Beads (PerkinElmer) to produce donor beadsthat mimic regulatory T cells expressing CTLA4 and CD3. Whether additionof 100 nmol/L of hUH02UL01/2C11-F760 can cause crosslinking betweendonor beads and acceptor beads in the presence of 50 μg/mL mouseCD3-acceptor beads (acceptor beads mimicking effector T cells) wasexamined. The condition in which hUH02UL01/2C11-F760 is not added to 100nmol/L of biotinylated mouse CTLA4; 50 μg/mL of AlphaScreen (registeredtrademark) Streptavidin-coated Donor Beads (PerkinElmer); and 50 μg/mLof mouse CD3-acceptor beads was used as the control in whichcrosslinking between each of the beads are not caused. All experimentswere performed three times. The results shown in FIG. 7-3 were obtainedfrom the experiments, and it was confirmed that even under the conditionwhere biotinylated mouse CD3 and mouse CTLA4 may be present on the samebead, the anti-mouse CTLA4/anti-mouse CD3 bispecific antibody crosslinksthe donor bead and acceptor bead as shown in FIG. 7-4.

The condition where biotinylated mouse CD3 and mouse CTLA4 are bound onthe donor beads was considered to be mimicking regulatory T cells(expressing CTLA4 and CD3), and mouse CD3-conjugated acceptor beads wasconsidered to be mimicking effector T cells (expressing CD3). It wasconfirmed that the anti-mouse CTLA4/anti-mouse CD3 bispecific antibodycrosslinks donor beads with acceptor beads under this condition as well,which suggests that even for regulatory T cells and effector T cells, ananti-CTLA4/anti-CD3 bispecific antibody may be able to form crosslinkingbetween the two cells in a similar manner.

[Reference Example 5] In Vivo Drug Efficacy Evaluation Using anAnti-Mouse CTLA4/Anti-Mouse CD3 Bispecific Antibody(hUH02UL01/2C11-F760) (Intratumoral Administration)

Whether the anti-mouse CTLA4 antibody hUH02hUL01-mFa55 purified andprepared by the method of Reference Example 2-1 and the anti-mouseCTLA4/anti-mouse CD3 bispecific antibody (hUH02UL01/2C11-F760) purifiedand prepared by the method of Reference Example 3-1 shows in vivo drugefficacy against a mouse colorectal cancer cell line was verified. 1×10⁶mouse colorectal cancer cell line CT26.WT cells (ATCC) weresubcutaneously transplanted into the right abdomen of BALB/c mice (JapanCharles River) to establish solid tumor. Ten days after thetransplantation, hUH02hUL01-mFa55 was administered at a dose of 200μg/mouse and hUH02UL01/2C11-F760 was administered at a dose of 100pig/mouse, intratumorally (i.t.) (n=2 for each group). The resultselucidated that hUH02UL01/2C11-F760 shows stronger antitumor effects incomparison to hUH02hUL01-mFa55, and shows remarkable antitumor effectsin vivo (FIG. 8). More specifically, the results suggested thathUH02UL01/2C11-F760 recognizes the surface antigens of regulatory Tcells (expressing CTLA4 and CD3) and effector T cells (expressing CD3),and causes crosslinking between the two cells in vivo.

[Reference Example 6] In Vivo Drug Efficacy Evaluation of an Anti-MouseCTLA4/Anti-Mouse CD3 Bispecific Antibody (hUH02UL01/2C11-F760)(Comparison Between Intratumoral Administration and IntravenousAdministration)

The anti-mouse CTLA4/anti-mouse CD3 bispecific antibody(hUH02UL01/2C11-F760) purified and prepared by the method of ReferenceExample 3-1 was assessed on whether it shows drug efficacy on the mousecolorectal cancer cell line CT26.WT-transplanted model described inReference Example 5 even when administered intravenously (i.v.). 1×10⁶CT26.WT cells (ATCC) were subcutaneously transplanted into the rightabdomen of BALB/c mice (Japan Charles River) to establish solid tumor.Eight days after the transplantation, hUH02UL01/2C11-F760 wasadministered at a dose of 100 μg/mouse intratumorally (i.t.) orintravenously (i.v.) (n=5 for each group). The results elucidated thathUH02UL01/2C11-F760 shows equivalent antitumor effects in bothintratumoral and intravenous administration, and shows antitumor effectsin vivo regardless of whether it is administered locally or systemically(FIG. 9).

[Reference Example 7] In Vitro Drug Efficacy Evaluation of an Anti-HumanCTLA4/Anti-Human CD3 Bispecific Antibody

(7-1) Expression and Purification of a Bispecific Antibody thatSpecifically Binds to Human CTLA4 and Human CD3

Genes encoding the variable regions of the anti-human CTLA4 antibodyMDX10-F760nN17 (the heavy chain variable region MDX10H is SEQ ID NO: 38,and the light chain variable region MDX10L is SEQ ID NO: 39) were eachinserted into human IgG1/kappa plasmids for expression in animals. Here,constant regions that have been modified so as to reduce binding to Fcγreceptors and to produce heterologous association of two heavy chainswere used (the heavy chain constant region F760nN17 is SEQ ID NO: 33,and the light chain constant region k0 is SEQ ID NO: 34).

Genes encoding the variable regions of the anti-human CD3 antibodyTR01H113-F760mG3P17 (the heavy chain variable region TR01H113 is SEQ IDNO: 40, and the light chain variable region L0011 is SEQ ID NO: 41) wereeach inserted into human IgG1/kappa plasmids for expression in animals.Here, constant regions that have been modified so as to reduce bindingto Fcγ receptors and to produce heterologous association of two heavychains were used (the heavy chain constant region F760nG3P17 is SEQ IDNO: 42, and the light chain constant region k0 is SEQ ID NO: 34).

Each of MDX10-F760nN17 and TR01H113-F760nG3P17 was expressed andpurified by the method shown in Reference Example 2. Each of thepurified homologous forms was mixed in the combination shown in Table 3,and the bispecific antibody of interest was produced by a method knownto those skilled in the art (WO2015/046467).

TABLE 3 No Name of clone Antibody 1 Antibody 2 1 MDX10//TR01H113MDX10-F760nN17 TR01H113-F760nG3P17

In addition, there are other techniques for forming bispecificantibodies. Examples include methods for antibody production usingassociation of antibody CH1 and CL, and association of VH and VL asdescribed in WO 2011/028952, WO2014/018572, and Nat Biotechnol. 2014February; 32(2): 191-8; methods using association of CH1 and CL or VHand VL, which are described in Proc Natl Acad Sci USA. 2011 Jul. 5;108(27): 11187-92, WO2009/080251, WO2009/080252, and WO2009/080253;methods for regulating association between antibody heavy chain CH3s,which are described in WO2012/058768 and WO2013/063702; methods thatutilize charge regulation of CH1 and CL, which are described inWO2006/106905; and methods that utilize charge regulation of VH and VL,which are described in WO2013/065708. The bispecific antibody ofinterest can be produced by applying the above-mentioned technologies toan anti-human CTLA4 antibody (the heavy chain variable region MDX10H isSEQ ID NO: 38, and the light chain variable region MDX10L is SEQ ID NO:39) and an anti-human CD3 antibody (the heavy chain variable regionTR01H113 is SEQ ID NO: 40, and the light chain variable region L0011 isSEQ ID NO: 41).

(7-2) In Vitro Cytotoxic Activity of an Anti-Human CTLA4/Anti-Human CD3Bispecific Antibody on Regulatory T Cells

Blood was collected using heparin from two healthy donors. Each bloodsample was diluted with HBSS (GIBCO) containing 5% FBS (MoregateBioTech), and then layered onto Ficoll-Paque Plus (GE healthcare). Thiswas centrifuged at 400×g for 30 minutes to separate the peripheral bloodmonocyte (PBMC) fraction. The obtained PBMCs were seeded into a 96-wellround-bottom plate (Corning) at 5×10⁵ cells/well using RPMI 1640(Nacalai Tesque) medium containing 10% FBS, and 100 Units/mLpenicillin—100 μg/mL Streptomycin (GIBCO).

The control antibody (the anti-KLH human IgG1 heavy chain variableregion IC17H is SEQ ID NO: 43, the light chain variable region IC17L isSEQ ID NO: 44, the heavy chain constant region hIgG1d is SEQ ID NO: 45,and the light chain constant region k0 is SEQ ID NO: 34) orMDX10/TR00H113 was diluted with the medium to each produce a finalconcentration of 0.1 μg/mL, 1 μg/mL, or 10 μg/mL, and added to thewells. The cells were cultured for seven days in a CO₂ incubator set at37° C. and 5% CO₂.

Seven days later, the cells were transferred to a V-bottom plate(Corning), and centrifuged at 400×g for five minutes to remove thesupernatant. The cells were resuspended in 100 μL of FcR blockingreagent (Miltenyi Biotec) diluted ten times with PBS containing 1% FBSand 2 mM EDTA (Sigma) (FACS buffer). After incubating at roomtemperature for ten minutes, 2.5 μL of PerCP-Cγ5.5 Mouse Anti-Human CD4(BD Pharmingen), 5 μL of PE Mouse Anti-Human CD25 (BD Pharmingen), and2.5 OL of PE-Cγ7 Mouse Anti-Human CD45RA (BD Pharmingen) were added toeach well. After incubating at 4° C. for one hour, 100 μL of FACS bufferwas added. Centrifugation was performed at 400×g for five minutes toremove the supernatant.

Based on the protocol of Intracellular Fixation and Permeabilizationbuffer set (eBioscience), Human FoxP3 buffer A was added 100 μL at atime, this was incubated at room temperature for ten minutes in thedark. Subsequently, centrifugation was performed at 400×g for fiveminutes to remove the supernatant. Permeabilization buffer was added 100μL at a time, this was incubated at room temperature for 30 minutes inthe dark. Next, 100 μL of FACS buffer was added, centrifugation wasperformed at 400×g for five minutes to remove the supernatant. Thiswashing procedure was performed one more time.

The cells were resuspended in 100 μL of FACS buffer, Alexa Fluor488Anti-Human FoxP3 (BioLegend) was added 5 μL at a time, this wasincubated at room temperature for 30 minutes in the dark. 100 μL of FACSbuffer was added, and centrifugation was performed at 400×g for fiveminutes to remove the supernatant. This washing procedure was performedone more time. The cells were resuspended in 200 μL of FACS buffer, andanalyzed on a FACS Cantoll flow cytometer (BD).

Expression analyses were performed using the FACSDiva Software (BD).CD4-positive cells were gated from the cell population subjected toanalysis and the expression of CD25 and CD45RA was analyzed. TheCD25^(high) CD45RA− fraction and the CD25-CD45RA+ fraction were regardedas the regulatory T cells (Treg) and effector T cells (Teff),respectively. Furthermore, the FoxP3highCD45RA− fraction and theFoxP3-CD45RA+ fraction were regarded as Treg and Teff, respectively. TheTeff/Treg ratio was calculated from the proportion of Treg and Teffpresent in CD4-positive cells.

The results of analyzing CD4-positive cells based on the expression ofCD25 and CD45RA are shown (FIG. 10-1). In Donor 1, treatment withMDX10/TR01H113 at 1 μg/mL and 10 μg/mL showed decrease in Treg. In Donor2, treatment at 1 μg/mL showed a decreasing trend in Treg, and treatmentat 10 μg/mL showed marked decrease in Treg (FIG. 10-2). In both donors,treatment with MDX 10/fR01H113 at 1 μg/mL and 10 μg/mL increased theTeff/Treg ratios (FIG. 10-3).

Furthermore, results of analyzing Treg based on the expression of FoxP3and CD45RA are shown in FIG. 11-1. Similarly to the analyses using CD25and CD45RA, treatment with MDX10/TR01H113 decreased Treg and increasedthe Teff/Treg ratios (FIGS. 11-2 and 11-3).

According to these examinations, TRAB (a bispecific antibody againstCTLA4 and CD3) which showed stronger antitumor effects with regard tocytotoxic activity against regulatory T cells expressing CTLA4 in vivoin mice, also showed strong cytotoxic activity against regulatory Tcells in vitro in humans; therefore, TRAB is expected to demonstratestrong antitumor effects towards cancer patients.

[Reference Example 8] Analysis of Surface Molecules Expressed in CellFractions (CD4⁺, CD25^(high), CD45RA⁻) that have been Reported to haveHigh Immune Response-Suppressing Function

Among the cells having immune response-suppressing functions, regulatoryT cells (Treg) in CD4-positive T cells calculated based on CD25 andCD45RA expression, that is the CD4+CD25^(high) CD45RA− cell fraction,have been reported to have high immune response-suppressing functions(Immunity, 2009, 30 (6), 899-911). Based on this information, a genewhich encodes a cell surface molecule showing significantly highexpression in the CD25^(high) CD45RA− cell fraction among theCD4-positive cells was identified using RNA-seq. As a result, amongCTLA4, PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137 (4-1BB),CD25, VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6, CD39, CD73,CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20, CD23, CD24, CD38,CD93, IgM, B220(CD45R), CD317, PD-L1, CD11b, Ly6G, ICAM-1, FAP, PDGFR,Podoplanin, and TIGIT, which are molecules expressed on the surface ofcells having immune response-suppressing function, nine molecules whichare CTLA4, TIM3, LAG3, CD137 (4-1BB), CD25, CCR5, CCR6, CD38, and TIGITwere found to be cell surface molecules highly expressed specifically inthe cell fractions (CD4+, CD25^(high), CD45RA−) that have been reportedto have high immune response-suppressing functions.

[Reference Example 9] In Vitro Drug Efficacy Evaluation of an Anti-HumanLAG3/Anti-Human CD3 Bispecific Antibody

(9-1) Expression and Purification of a Bispecific Antibody thatSpecifically Binds to Human LAG3 and Human CD3

Genes encoding the variable regions of the anti-human LAG3 antibody25F7-F760nN17 (the heavy chain variable region 25F7H is SEQ ID NO: 46,and the light chain variable region 25F7L is SEQ ID NO: 47) were eachinserted into human IgG1/kappa plasmids for expression in animals. Here,constant regions that have been modified so as to reduce binding to Fcγreceptors and to produce heterologous association of two heavy chainswere used (the heavy chain constant region F760nN17 is SEQ ID NO: 33,and the light chain constant region k0 is SEQ ID NO: 34).

Genes encoding the variable regions of the anti-human CD3 antibodyTR01H113-F760nG3P17 (the heavy chain variable region TR01H113 is SEQ IDNO: 40, and the light chain variable region L0011 is SEQ ID NO: 41) wereeach inserted into human IgG1/kappa plasmids for expression in animals.Here, constant regions that have been modified so as to reduce bindingto Fcγ receptors and to produce heterologous association of two heavychains were used (the heavy chain constant region F760nG3P17 is SEQ IDNO: 42, and the light chain constant region k0 is SEQ ID NO: 34).

Each of 25F7-F760nN17 and TR01H113-F760nG3P17 was expressed and purifiedby the method shown in Reference Example 2. Each of the purifiedhomologous forms was mixed in the combination shown in Table 4, and thebispecific antibody of interest was produced by a method known to thoseskilled in the art (WO2015/046467).

TABLE 4 No Name of clone Antibody 1 Antibody 2 1 25F7//TR01H11325F7-F760nN17 TR01H113-F760nG3P17

(9-2) In Vitro Cytotoxic Activity of an Anti-Human LAG3/Anti-Human CD3Bispecific Antibody on Regulatory T Cells

Blood was collected using heparin from healthy donors. Each blood samplewas diluted with PBS and then layered together with Ficoll-Paque Plus(GE healthcare) in a Leucosep tube (greiner bio-one). This wascentrifuged at 1000×g for 10 minutes to separate the peripheral bloodmonocyte (PBMC) fraction. The obtained PBMCs were seeded into a 96-wellround bottom plate (Corning) at 1×10⁶ cells/well using RPMI 1640(Nacalai Tesque) medium containing 10% FBS, and 100 Units/mLpenicillin—100 μg/mL Streptomycin (GIBCO).

TRAB (25F7/f/R01H113) was diluted with the medium at a finalconcentration of 1 μg/mL or 10 μg/mL, and added to the wells. The cellswere cultured for four or six days in a CO₂ incubator set at 37° C. and5% CO2.

Four or six days later, the cells were transferred to tubes for FACSanalysis, and centrifuged at 400×g for five minutes to remove thesupernatant. Cell WASH (BD Biosciences) containing 0.2% BSA (Wako) wasprepared, and this was used as the FACS Buffer. For complete removal ofmedium components, washing was performed by adding 2 mL of FACS Bufferto the cells from which the supernatant was removed and performing thecentrifugation again at 400×g for five minutes to remove thesupernatant.

FcR blocking reagent (Miltenyi Biotec) diluted ten-fold with the FACSBuffer, to which 1/1000 volume of eFluor780 (eBioscience) for stainingdead cells was added, was prepared and used as the Staining Buffer.Solution produced by adding 5 μL of PerCP Mouse Anti-Human CD4 (BDPharmingen), 2.5 μL of PE-Cy™ 7 Mouse Anti-Human CD45RA (BD Pharmingen),and 5 μL of PE Mouse Anti-Human CD25 to 50 μL of the Staining Buffer wasplaced into each tube. After incubation at 4° C. for one hour, 2 mL ofFACS buffer was added, and the centrifugation was performed at 400×g forfive minutes to remove the supernatant. Then, as a washing procedure, anadditional 2 mL of FACS buffer was added, and the centrifugation wasperformed at 400×g for five minutes to remove the supernatant. The cellswere resuspended in 400 μL of FACS buffer and analyzed on a FACSVerse™flow cytometer (BD).

Expression analysis was carried out using the FACSDiva Software (BD).CD4-positive cells were gated from the cell population subjected foranalysis, from which dead cells had been removed, and the expression ofCD25 and CD45RA was analyzed. The CD25^(high) CD45RA− fraction and theCD25− CD45RA+ fraction were regarded as regulatory T cells (Treg) andeffector T cells (Teff), respectively. The Teff/Treg ratio wascalculated from the proportion of Treg and Teff present in CD4-positivecells.

The results of analyzing CD4-positive cells based on the expression ofCD25 and CD45RA are shown (FIG. 13). Treatment with TRAB (25F7/TR01H113)at 1 μg/mL and 10 μg/mL showed decrease in Treg in a TRAB antibodydose-dependent manner, both at four and six days after the treatment(FIG. 14). Treatment with TRAB (25F7/TR01H1113) at 1 μg/mL and 10 μg/mLincreased the Teff/Treg ratios (FIG. 15).

[Reference Example 10] In Vitro Drug Efficacy Evaluation of anAnti-Human OX40/Anti-Human CD3 Bispecific Antibody

(10-1) Expression and Purification of a Bispecific Antibody thatSpecifically Binds to Human OX40 and Human CD3

Genes encoding the variable regions of the anti-human OX40 antibody12H3-F760nN17 (the heavy chain variable region 12H3VH is SEQ ID NO: 48,and the light chain variable region 12H3VL is SEQ ID NO: 49) were eachinserted into human IgG1/kappa plasmids for expression in animals. Here,constant regions that have been modified so as to reduce binding to Fcγreceptors and to produce heterologous association of two heavy chainswere used (the heavy chain constant region F760nN17 is SEQ ID NO: 33,and the light chain constant region k0 is SEQ ID NO: 34).

Genes encoding the variable regions of the anti-human CD3 antibodyTR01H113-F760nG3P17 (the heavy chain variable region TR01H113 is SEQ IDNO: 40, and the light chain variable region L0011 is SEQ ID NO: 41) wereeach inserted into human IgG1/kappa plasmids for expression in animals.Here, constant regions that have been modified so as to reduce bindingto Fcγ receptors and to produce heterologous association of two heavychains were used (the heavy chain constant region F760nG3P17 is SEQ IDNO: 42, and the light chain constant region k0 is SEQ ID NO: 34).

Each of 12H3-F760nN17 and TR01H113-F760nG3P17 was expressed and purifiedby the method shown in Reference Example 2. Each of the purifiedhomologous forms was mixed in the combination shown in Table 5, and thebispecific antibody of interest was produced by a method known to thoseskilled in the art (WO2015/046467).

TABLE 5 No Name of clone Antibody 1 Antibody 2 1 12H3//TR01H11312H3-F760nN17 TR01H113-F760nG3P17

(10-2) In Vitro Cytotoxic Activity of an Anti-Human OX40/Anti-Human CD3Bispecific Antibody on Regulatory T Cells

Blood was collected using heparin from two healthy donors. Each bloodsample was diluted with PBS and then layered together with Ficoll-PaquePlus (GE healthcare) in a Leucosep tube (greiner bio-one). Thecentrifugation was performed at 1000×g for 10 minutes to separate theperipheral blood monocyte (PBMC) fraction. The obtained PBMCs wereseeded into a 96-well round bottom plate (Corning) at 1×10⁶ cells/wellusing RPMI 1640 (Nacalai Tesque) medium containing 10% FBS, and 100Units/mL penicillin—100 μg/mL Streptomycin (GIBCO).

TRAB (12H3/TR01H113) was diluted with the medium at a finalconcentration of 1 μg/mL or 10 μg/mL, and was added to the wells. Thecells were cultured for seven days in a CO₂ incubator set at 37° C. and5% CO₂.

Seven days later, the cells were transferred to tubes for FACS analysis,and centrifuged at 400×g for five minutes to remove the supernatant.Cell WASH (BD Biosciences) containing 0.2% BSA (Wako) was prepared andused as the FACS Buffer. For complete removal of medium components,washing was performed by adding 2 mL of FACS Buffer to the cells fromwhich the supernatant was removed and performing the centrifugationagain at 400×g for five minutes to remove the supernatant.

FcR blocking reagent (Miltenyi Biotec) diluted ten-fold with the FACSBuffer, to which 1/1000 volume of eFluor780 (eBioscience) for stainingdead cells was added, was prepared and used as the Staining Buffer.Solution produced by adding 5 μL of PerCP Mouse Anti-Human CD4 (BDPharmingen), 2.5 μL of PE-CyM7 Mouse Anti-Human CD45RA (BD Pharmingen),and 5 μL of PE Mouse Anti-Human CD25 to 50 μL of the Staining Buffer wasplaced into each tube. After incubation at 4° C. for one hour, 2 mL ofFACS Buffer was added, and the centrifugation was performed at 400×g forfive minutes to remove the supernatant. Then, as a washing procedure, 2mL of FACS buffer was further added, and the centrifugation wasperformed at 400×g for five minutes to remove the supernatant. The cellswere resuspended in 400 μL of FACS buffer and analyzed on a FACSVerse™flow cytometer (BD).

Expression analysis was carried out using the FACSDiva Software (BD).CD4-positive cells were gated from the cell population subjected toanalysis, from which dead cells had been removed, and the expression ofCD25 and CD45RA was analyzed. The CD25^(high) CD45RA− fraction and theCD25− CD45RA+ fraction were regarded as regulatory T cells (Treg) andeffector T cells (Teff), respectively. The Teff/Treg ratio wascalculated from the proportion of Treg and Teff present in CD4-positivecells.

The results of analyzing CD4-positive cells based on the expression ofCD25 and CD45RA are shown (FIG. 16). Treatment of PBMCs derived fromboth donors with TRAB (12H3/TR01H113) at 1 μg/mL and 10 μg/mL showeddecrease in Treg in a TRAB antibody dose-dependent manner (FIG. 17).Treatment with TRAB (12H3/TR01H113) at 1 μg/mL and 10 μg/mL increasedthe Teff/Treg ratios (FIG. 18).

[Reference Example 11] ADCC Activity of a Test Antibody Using MouseFcgR4-Expressing Human NK Cell Line NK-92 as the Effector Cells

Regarding anti-mouse CTLA4 antibodies, antibody concentration-dependentADCC activities of test antibodies were measured, using the mouseFcgR4-expressing human NK cell line NK-92 (hereinafter referred to asmFcgR4-NK92) as effector cells by following the method described below.

(1) Preparation of mFcgR4-NK92 Solution

After washing mFcgR4-NK92 with RPMI-1640 (nacalai tesque) containing 10%FBS (hereinafter referred to as 10% FBS/RPMI), the cells were suspendedin 10% FBS/RPMI at a cell density of 4×10⁵ cells/ml. This cellsuspension solution was used as the mFcgR4-NK92 solution in thesubsequent experiments.

(2) Preparation of Target Cells

To 2×10⁶ CHO/mouse CTLA4 cells which are CHO cells forced to expressmouse CTLA4, 3.7 MBq of Cr-51 was added. The Cr-51-added cells wereincubated in a 5% carbon dioxide gas incubator at 37° C. for one hour,then washed three times with 10% FBS/RPMI, and then suspended in 10%FBS/RPMI at a cell density of 2×10⁵ cells/ml. The cell suspension wasused as the target cells in the subsequent experiments.

(3) Chromium-Release Assay (ADCC Activity)

The ADCC activities were evaluated from the specific chromium releaserate according to the chromium release method. First, antibody solutionsprepared at each concentration (0, 0.04, 0.4, 4, and 40 μg/ml) wereadded to a 96-well U-bottomed plate at 50 μl per well. Next, the targetcells prepared in (2) were seeded at 50 μl per well (1×10⁴ cells/well).Furthermore, 10% FBS/RPMI was added at 50 Ipl per well, and the platewas allowed to stand at room temperature for 15 minutes. The mFcgR4-NK92solution prepared in (1) was added at 50 μl per well (2×10⁴ cells/well),the plate was left to stand in a 5% carbon dioxide gas incubator at 37°C. for four hours, and resultant was centrifuged. The radioactivity of100 μl of culture supernatant in each well of the plate was measuredusing a gamma counter. The specific chromium release rate was determinedbased on the following equation.

Chromium release rate (%)=(A−C)×100/(B−C)

In this equation, “A” represents the mean value of radioactivity (cpm)of 100 μl of culture supernatant in each well; “B” represents the meanvalue of radioactivity (cpm) of 100 μl of culture supernatant in a wellwhere 50 μl of a 4% NP-40 aqueous solution (Nonidet P-40, NacalaiTesque) and 100 μl of 10% FBS/RPMI had been added to the target cells;and “C” represents the mean value of radioactivity (cpm) of 100 μl ofculture supernatant in a well where 150 μl of 10% FBS/RPMI had beenadded to the target cells. The examinations were performed in duplicateand the mean value for the specific chromium release rate (%) of thetest antibody was calculated.

[Reference Example 12] Measurement of Cytotoxic Activity of a TestAntibody Using Mouse Splenocytes as Effector Cells

Regarding the anti-mouse CTLA4/anti-mouse CD3 bispecific antibody(hUH02UL01/2C11-F760), antibody concentration-dependent cytotoxicactivity of the test antibody was measured using mouse splenocytes aseffector cells, and by following the method described below.

(1) Preparation of Mouse Splenocyte Solution

Ten mL of 10% FBS/RPMI was added to the spleen excised from a BALB/cmouse. The spleen was sliced into small pieces and passed through a cellstrainer. After centrifugation (at 2,150 rpm for ten minutes at roomtemperature), a hemolysis procedure was performed using a MouseErythrocyte Lysing Kit (R&D Systems). After washing once with 10%FBS/RPMI, the cells were suspended in 10% FBS/RPMI at a cell density of6×10⁶ cells/ml. The cell suspension was used as the mouse splenocytesolution in the subsequent experiments.

(2) Cytotoxic Activity Evaluation Assay

Cytotoxic activity was evaluated by cell proliferation inhibition rateusing the xCELLigence Real-Time Cell Analyzer (Roche Diagnostics K.K.).CHO/mCTLA4 which is CHO forced to express mouse CTLA4 was used as thetarget cells. The cells were suspended in CHO-S-SFM II (Lifetechnologies) containing 10% FBS, and were seeded in aliquots of 100 μlinto an E-Plate 96 plate (Roche Diagnostics K.K.) at 5×10³ cells/well.Measurement of living cells was started using the xCELLigence Real-TimeCell Analyzer. On the following day, the plate was removed from thexCELLigence Real-Time Cell Analyzer, and 50 μl of the respectiveantibodies prepared at each concentration (0.04, 0.4, 4, or 40 μg/ml)were added to the plate. After allowing this to stand at roomtemperature for 15 minutes, 50 μl of the mouse splenocyte solutionprepared in (1) was added (3×10⁵ cells/well). By setting the plate intothe xCELLigence Real-Time Cell Analyzer again, measurement of livingcells was started. The reaction was carried out in a 5% carbon dioxidegas incubator at 37° C., and from the Cell Index value obtained 72 hoursafter addition of the mouse splenocyte solution, the cell proliferationinhibition rate (%) was determined using the following equation. TheCell Index value used in the calculation was a normalized value wherethe Cell Index value immediately before antibody addition was defined as1.

Cell proliferation inhibition rate (%)=(A−B)×100/(A−1)

“A” represents the mean value of the Cell Index values in wells withoutantibody addition (containing only the target cells and human PBMC), and“B” represents the mean value of the Cell Index values in each well. Theexaminations were performed in duplicate.

[Reference Example 13] Production of an Anti-Mouse PD1 Antibody

An anti-mouse PD1 antibody (mPD1F2-mFa31) was produced. Heavy-chainvariable region mPD1F2VH (SEQ ID NO: 50) and light-chain variable regionmPD1F2VL (SEQ ID NO: 51) were used, and mouse heavy-chain constantregion mFa31 (SEQ ID NO: 52) and wildtype mouse light-chain constantregion mk1 (SEQ ID NO: 31) were used as the constant regions. In thatcase, the mouse heavy-chain constant region used was one which had beenmodified to decrease binding to Fcγ receptors.

The anti-mouse PD1 antibody was expressed using the following method.Cells of human embryonic kidney cell-derived FreeStyle 293-F strain(Invitrogen) were suspended and inoculated in the FreeStyle 293Expression Medium (Invitrogen) at a cell density of 1.33×10⁶ cells/mL.Prepared plasmids were introduced into the cells by a lipofectionmethod. The cells were cultured for four days in a CO₂ incubator (37°C., 8% CO₂, 90 rpm). From the culture supematants, antibodies werepurified using Protein A Sepharose™ Fast Flow (Amersham Biosciences) bya method known to those skilled in the art. Absorbances at 280 nm of thepurified antibody solutions were measured using a spectrophotometer.Concentrations of the purified antibodies were calculated from thedetermined values using an extinction coefficient calculated by the PACEmethod (Protein Science (1995) 4, 2411-2423).

[Reference Example 14] Production of an Anti-Mouse CTLA4/Anti-Mouse CD3Bispecific Antibody and an Anti-Human GPC3/Anti-Mouse CD3 BispecificAntibody (1) Production of an Anti-Mouse CTLA4/Anti-Mouse CD3 BispecificAntibody

An anti-mouse CTLA4/anti-mouse CD3 bispecific antibody (mCTLA4/mCD3),which is a combination of an anti-mouse CTLA4 antibody and an anti-mouseCD3 antibody, was produced. Heavy chain variable region hUH02 (SEQ IDNO: 28) and light chain variable region hUL01 (SEQ ID NO: 29) were usedon the anti-mouse CTLA4 arm. In that case, the constant regions usedwere heavy chain constant region mF18mN4 (SEQ ID NO: 53), which had beenmodified to decrease binding to Fcγ receptors and enable heterologousassociation between the two heavy chains, and light chain constantregion mk 1 (SEQ ID NO: 31). Furthermore, heavy chain variable region2C11VH (SEQ ID NO: 35) and light chain variable region 2C11VL (SEQ IDNO: 36) were used on the anti-mouse CD3 arm. In that case, the constantregions used were heavy chain constant region mF18mP4 (SEQ ID NO: 54),which had been modified to decrease binding to Fcγ receptors and enableheterologous association between the two heavy chains, and light chainconstant region mk1 (SEQ ID NO: 31).

(2) Production of an Anti-Human GPC3/Anti-Mouse CD3 Bispecific Antibody

An anti-human GPC3/anti-mouse CD3 bispecific antibody (hGPC3/mCD3),which is a combination of an anti-human GPC3 antibody and an anti-mouseCD3 antibody, was produced. Heavy chain variable region H0000 (SEQ IDNO: 55) and light chain variable region GL4 (SEQ ID NO: 56) were used onthe anti-human GPC3 arm. In that case, the constant regions used wereheavy chain constant region mF18mN4 (SEQ ID NO: 53), which had beenmodified to decrease binding to Fcγ receptors and enable heterologousassociation between the two heavy chains, and light chain constantregion mk 1 (SEQ ID NO: 31). Furthermore, heavy chain variable region2C11VH (SEQ ID NO: 35) and light chain variable region 2C11VL (SEQ IDNO: 36) were used on the anti-mouse CD3 arm. In that case, the constantregions used were heavy chain constant region mF18mP4 (SEQ ID NO: 54),which had been modified to decrease binding to Fcγ receptors and enableheterologous association between the two heavy chains, and light chainconstant region mk1 (SEQ ID NO: 35).

(3) Antibody Expression

The antibodies of (1) and (2) above were expressed using the followingmethod.

Cells of human embryonic kidney cell-derived FreeStyle 293-F strain(Invitrogen) were suspended and inoculated in the FreeStyle 293Expression Medium (Invitrogen) at a cell density of 1.33×10⁶ cells/mL.Prepared plasmids were introduced into the cells by a lipofectionmethod. The cells were cultured for four days in a CO₂ incubator (37°C., 8% CO₂, 90 rpm). From the culture supematants, antibodies werepurified using the Hi Trap™ Protein G HP column (GE Healthcare) by amethod known to those skilled in the art. Absorbances at 280 nm of thepurified antibody solutions were measured using a spectrophotometer.Concentrations of the purified antibodies were calculated from thedetermined values using an extinction coefficient calculated by the PACEmethod (Protein Science (1995) 4: 2411-2423).

Each of the purified homologous forms was mixed in the combinationsshown in Table 6, and the bispecific antibodies of interest wereproduced by a method known to those skilled in the art that utilizesdifferences in the charges of the constant regions (WO2015/046467).

TABLE 6 No Name of clone Antibody 1 Antibody 2 1 mCTLA4//mCD3hUH02/hUL01-mF18mN4 2C11-mF18mP4 2 hGPC3//mCD3 H0000/GL4-mF18mN42C11-mF18mP4

[Reference Example 15] Production of an Anti-Human GPC3/Anti-Mouse CD137Bispecific Antibody

GPC3 ERY22-3-1D8, which is an anti-human GPC3/anti-mouse CD137bispecific antibody, was produced. The H chains of GPC3 ERY22-3-1D8comprise two types of H chains: GC33(2)H-GldKnHSG3 (SEQ ID NO: 57) asthe anti-human GPC3 arm H-chain constant region gene, prepared byintroducing the H435R modification for simplifying purification known tothose skilled in the art; and 1D8VH-GldHIS (SEQ ID NO: 58) as theanti-mouse CD137 arm H chain constant region gene. As the L chains ofGPC3 ERY22-3-1D8, GC33(2)L-k0 (SEQ ID NO: 59) and 1D8VL-k0 (SEQ ID NO:60) were used on the anti-human GPC3 arm and the anti-mouse CD137 arm,respectively, to obtain the bispecific antibody of interest.

The antibody was expressed using the following method. Cells of humanembryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) weresuspended and inoculated in the FreeStyle 293 Expression Medium(Invitrogen) at a cell density of 1.33×10⁶ cells/mL. Prepared plasmidswere introduced into the cells by a lipofection method. The cells werecultured for four days in a CO₂ incubator (37° C., 8% CO₂, 90 rpm). Theobtained culture supernatant was added to a MabSelect SuRe column (GEHealthcare), and the column was washed, followed by elution with 50 mMacetic acid. The antibody-containing fractions were added to a HisTrapHP column (GE Healthcare) or a Ni Sepharose FF column (GE Healthcare),and the column was washed, followed by elution with imidazole. Theantibody-containing fractions were concentrated using an ultrafiltrationmembrane. Then, the concentrated solution was added to a Superdex 200column (GE Healthcare). Only the monomeric antibodies in the eluate werecollected to obtain the purified antibodies.

TABLE 7 H-chain L-chain H-chain L-chain Name of clone gene 1 gene 1 gene2 gene 2 GPC3 ERY22- GC33 (2) GC33 (2) 1D8VH- 1D8VL- 3-1D8 H-G1dKnHSG3L-k0 G1dHIS k0

[Example 1] Preparation of Syngeneic Tumor Cell-Engrafted Mouse Models(1) Cell Lines

CT26.WT cells (ATCC No.: CRL-2638) or LL/2 (LLC1) cells (ATCC No.:CRL-1642) were used.

CT26/hGPC3 cell line that expresses human GPC3 in high levels wasobtained by integrating the human GPC3 gene into the chromosome of themouse colorectal cancer cell line CT26.WT by a method well known tothose skilled in the art. The expression level of human GPC3(2.3×10⁵/cell) was determined using the QIFI kit (Dako) by themanufacturer's recommended method.

CT26.WT cells were maintained and passaged in RPMI-1640 medium(manufactured by SIGMA) containing 10% FBS (manufactured by BOVOGEN),0.5% D-glucose (manufactured by SIGMA), 1 mM Sodium Pyruvate(manufactured by ThermoFisher), and 10 mM HEPES (manufactured by SIGMA).The CT26/hGPC3 cell line was maintained and passaged in 10%FBS-containing RPMI-1640 (SIGMA, Cat. No. R8758) supplemented with 1 mMSodium Pyruvate (Gibco, Cat. No. 11360-070), 10 mM HEPES (Sigma, Cat.No. H0887), 0.45% D-glucose (Sigma, Cat. No. G8769), and 200 μg/mL G-418(Nacalai tesque, Cat. No. 09380-44).

The human GPC3 gene was integrated into the chromosome of the mouse lungcancer cell line LLC1 by a method well known to those skilled in the artto obtain an LLC1/hGPC3 cell line that expresses human GPC3 in highlevels. The LL/2 (LLC1) cells and the LLC1/hGPC3 cell line weremaintained and passaged in D-MEM (high glucose) medium (manufactured bySIGMA) containing 10% FBS (manufactured by BOVOGEN).

(2) Syngeneic Tumor Cell Engrafted Mouse Models

BALB/cA mice and C57BL/6N mice were purchased from Charles RiverLaboratories Japan, Inc. The CT26.WT cells or the CT26/hGPC3 cells weretransplanted subcutaneously to BALB/cA mice, and the LL/2 (LLC1) cellsor the LLC1/hGPC3 cells were transplanted subcutaneously to C57BL/6Nmice. Models were established when the average volume of thetransplanted tumors reached approximately 100 mm³ to 200 mm³.

The volume of the transplanted tumor was calculated using the followingequation:

Tumor volume (mm³)=major axis (mm)×minor axis (mm)×minor axis (mm)/2

[Example 2] Preparation of Pharmaceutical Agents to be Administered (1)The Pharmaceutical Agents to be Administered for Use in Test of CombinedUse of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific Monoclonal Antibodyand Gemcitabine

As the pharmaceutical agents to be administered for the combined usetest, the anti-mouse CTLA4/anti-mouse CD3 bispecific monoclonal antibody(mCTLA4/mCD3) was prepared at 0.125 mg/mL in PBS (−), and a pyrimidineanalog (antimetabolite), gemcitabine (Gemzar: Eli Lilly Japan) wasprepared at 12 mg/mL in physiological saline solution (OtsukaPharmaceutical).

(2) The Pharmaceutical Agents to be Administered for Use in Test ofCombined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific MonoclonalAntibody and Cisplatin

As the pharmaceutical agents to be administered for the combined usetest, the anti-mouse CTLA4/CD3 bispecific monoclonal antibody(mCTLA4/mCD3) was prepared at 0.5 mg/mL in PBS (−), and, for a platinumcompound, cisplatin (Briplatin: Bristol-Myers Co.), its stock solution(10 mg/20 mL) was administered as it was.

(3) The Pharmaceutical Agents to be Administered for Use in Examinationof Combined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 BispecificMonoclonal Antibody and an Anti-Mouse PD1 Monoclonal Antibody

As the pharmaceutical agents to be administered for the combined usetest, PBS (−) was used to prepare the anti-mouse CTLA4/CD3 bispecificmonoclonal antibody (mCTLA4/mCD3) at 0.5 mg/mL and an immune checkpointinhibitor, the anti-mouse PD1 monoclonal antibody (mPD1F2-mFa31) at 1.5mg/mL.

(4) The Pharmaceutical Agents to be Administered for Use in Test ofCombined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific MonoclonalAntibody and an Anti-Human GPC3/Anti-Mouse CD3 Bispecific MonoclonalAntibody

As the pharmaceutical agents to be administered for the combined usetest, PBS (−) was used to prepare the anti-mouse CTLA4/anti-mouse CD3bispecific monoclonal antibody (mCTLA4/mCD3) at 0.5 mg/mL and theanti-human GPC3/anti-mouse CD3 bispecific monoclonal antibody(hGPC3/mCD3) at 0.5 mg/mL.

(5) The Pharmaceutical Agents to be Administered for Use in Test ofCombined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific MonoclonalAntibody and an Anti-Mouse CD137/Anti-Human GPC3 Bispecific Antibody

As the pharmaceutical agents to be administered for the combined usetest, the anti-mouse CD137/human GPC3 bispecific antibody (GPC3ERY22-3-1D8) and the anti-mouse CTLA4/anti-mouse CD3 bispecific antibody(mCTLA4/mCD3) were prepared at 0.05 mg/mL in 0.05% Tween 20/PBS.

[Example 3] Test of Combined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3Bispecific Monoclonal Antibody and Other Anticancer Agents 1-(1) Test ofCombined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific MonoclonalAntibody and Gemcitabine

To evaluate the effects of combined use of the anti-mouseCTLA4/anti-mouse CD3 bispecific monoclonal antibody (mCTLA4/mCD3) andgemcitabine, the CT26.WT cell-transplanted model was used and giventhrough the tail vein gemcitabine at 120 mg/kg eight days aftertransplantation and mCTLA3/mCD3 at 25 μg/mouse nine days aftertransplantation.

1-(2) the Pharmaceutical Agents to be Administered for Use in Test ofCombined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific MonoclonalAntibody and Cisplatin

To evaluate the effects of combined use of the anti-mouseCTLA4/anti-mouse CD3 bispecific monoclonal antibody (mCTLA4/mCD3) andcisplatin, the LL/2 (LLC1) cell-transplanted model was used and giventhrough the tail vein cisplatin at 5 mg/kg ten days aftertransplantation and mCTLA4/mCD3 at 100 μg/mouse eleven days aftertransplantation.

1-(3) the Pharmaceutical Agents to be Administered for Use in Test ofCombined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific MonoclonalAntibody and an Anti-Mouse PD1 Monoclonal Antibody

To evaluate the effects of combined use of the anti-mouse CTLA4/CD3bispecific monoclonal antibody (mCTLA4/mCD3) and the anti-mouse PD1monoclonal antibody (mPD1F2-mFa31), the LL/2 (LLC1) cell-transplantedmodel was used and, seven days after transplantation, given through thetail vein mCTLA4/mCD3 at 100 μg/mouse and mPD1F2-mFa31 at 300 μg/mouse.

1-(4) the Pharmaceutical Agents to be Administered for Use in Test ofCombined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3 Bispecific MonoclonalAntibody and an Anti-Human GPC3/Anti-Mouse CD3 Bispecific MonoclonalAntibody

To evaluate the effects of combined use of the anti-mouse CTLA4/CD3bispecific monoclonal antibody (mCTLA4/mCD3) and the anti-humanGPC3/anti-mouse CD3 bispecific monoclonal antibody (hGPC3/mCD3), theLLC1/hGPC3 cell-transplanted model was used and, eight days aftertransplantation, given mCTLA4/mCD3 and hGPC3/mCD3 each at 100 μg/mousethrough the tail vein.

1-(5) Test of Combined Use of an Anti-Mouse CTLA4/Anti-Mouse CD3Bispecific Monoclonal Antibody and an Anti-Mouse CD137/Anti-Human GPC3Bispecific Antibody

To evaluate the effects of combined use of the anti-mouseCTLA4/anti-mouse CD3 bispecific monoclonal antibody (mCTLA4/mCD3) andthe anti-mouse CD137/anti-human GPC3 bispecific monoclonal antibody(GPC3 ERY22-3-1D8), the CT26/hGPC3 cell-transplanted model was used and,14, 17, and 21 days after transplantation, given mCTLA4/mCD3 and GPC3ERY22-3-1D8 each at 500 μg/kg through the tail vein.

The details of the pharmaceutical agent treatments described above in1-(1) to 1-(5) are shown in Tables 8 to 12.

TABLE 8 CT26.WT cell-transplanted model (anti-mouse CTLA4/anti-mouse CD3antibody and gemcitabine) Number of Pharmaceutical Method of Day ofGroup animals agent Dose administration administration 1 5 PBS (—) —Tail vein 8 days after transplantation 2 5 mCTLA4//mCD3  25 μg/mouseTail vein 9 days after transplantation 3 5 gemcitabine 120 mg/kg Tailvein 8 days after transplantation 4 5 mCTLA4//mCD3  25 μg/mouse Tailvein 9 days after transplantation gemcitabine 120 mg/kg Tail vein 8 daysafter transplantation

TABLE 9 LL/2 (LLC1) cell-transplanted model (anti-mouse CTLA4/anti-mouseCD3 antibody and cisplatin) Number of Pharmaceutical Method of Day ofGroup animals agent Dose administration administration 1 5 PBS (—) —Tail vein 10 days after transplantation 2 5 mCTLA4//mCD3 100 μg/mouseTail vein 11 days after transplantation 3 5 cisplatin  5 mg/kg Tail vein10 days after transplantation 4 5 mCTLA4//mCD3 100 μg/mouse Tail vein 11days after transplantation cisplatin  5 mg/kg Tail vein 10 days aftertransplantation

TABLE 10 LL/2 (LLC1) cell-transplanted model (anti-mouseCTLA4/anti-mouse CD3 antibody and anti-mouse PD1 antibody) Number ofMethod of Day of Group animals Pharmaceutical agent Dose administrationadministration 1 5 PBS (—) — Tail vein 7 days after transplantation 2 5mCTLA4//mCD3 100 μg/mouse Tail vein 7 days after transplantation 3 5mPD1F2-mFa31 300 μg/mouse Tail vein 7 days after transplantation 4 5mCTLA4//mCD3 100 μg/mouse Tail vein 7 days after transplantationmPD1F2-mFa31 300 μg/mouse Tail vein 7 days after transplantation

TABLE 11 LLC1/hGPC3 cell-transplanted model (anti-mouse CTLA4/anti-mouseCD3 antibody and anti-human GPC3/anti-mouse CD3 antibody) Number ofMethod of Day of Group animals Pharmaceutical agent Dose administrationadministration 1 5 PBS (—) — Tail vein 8 days after transplantation 2 5mCTLA4//mCD3 100 μg/mouse Tail vein 8 days after transplantation 3 5hGPC3//mCD3 100 μg/mouse Tail vein 8 days after transplantation 4 5mCTLA4//mCD3 100 μg/mouse Tail vein 8 days after transplantationhGPC3//mCD3 100 μg/mouse Tail vein 8 days after transplantation

TABLE 12 CT26/hGPC3 cell-transplanted model (anti-mouse CTLA4/anti-mouseCD3 antibody and anti-mouse CD137/anti-human GPC3 antibody) Number ofPharmaceutical Method of Group animals agent Dose administration Day ofadministration 1 5 0.05% Tween 20 — Tail vein 14, 17, and 21 days aftertransplantation PBS 2 5 mCTLA4//mCD3 500 μg/kg Tail vein 14, 17, and 21days after transplantation 3 5 GPC3 ERY22-3- 500 μg/kg Tail vein 14, 17,and 21 days after transplantation 1D8 4 5 mCTLA4//mCD3 500 μg/kg Tailvein 14, 17, and 21 days after transplantation GPC3 ERY22-3- 500 μg/kgTail vein 14, 17, and 21 days after transplantation 1D8

2. Evaluation of Antitumor Effects in Combined Use Tests

Antitumor effects were evaluated from the tumor volumes calculated usingthe equation shown in (2) of Example 1. SAS preclinical package (SASInstitute Inc.) was used for statistical analyses. The evaluationresults are shown in FIGS. 19 to 23. As a result of the evaluation, itwas shown that tumor growth-suppressing effects of the anti-mouseCTLA4/anti-mouse CD3 bispecific monoclonal antibody were increased byusing in combination with any one of gemcitabine, cisplatin, theanti-mouse PD1 antibody, the anti-human GPC3/anti-mouse CD3 antibody,and the anti-mouse CD137/anti-human GPC3 antibody.

1. A method for inducing cytotoxicity, for suppressing cellproliferation, for inhibiting cell proliferation, for activating immuneresponse, for treating cancer, or for preventing cancer in anindividual, each method comprising administering an effective amount ofan anticancer agent and an effective amount of a first antigen-bindingmolecule that comprises: (1) a domain that binds to a molecule expressedon the surface of a cell having an immune response-suppressing function;and (2) a T cell receptor complex-binding domain, and inhibits theimmune response-suppressing activity of the cell having an immuneresponse suppressing function.
 2. (canceled)
 3. The method of claim 1,wherein the first antigen-binding molecule and the anticancer agent areadministered simultaneously or sequentially.
 4. The method of claim 1,wherein the first antigen-binding molecule further comprises anFcRn-binding domain.
 5. The method of claim 1, wherein the FcRn-bindingdomain is an antibody Fc region having decreased Fcγ receptor-bindingactivity.
 6. The method of claim 1, wherein the first antigen-bindingmolecule is a multispecific antibody.
 7. The method of claim 1, whereinthe cell having an immune response-suppressing function is a regulatoryT cell or an exhausted T cell.
 8. The method of claim 1, wherein themolecule expressed on the surface of the cell having an immuneresponse-suppressing function is any molecule selected from among CTLA4,PD1, TIM3, LAG3, CD244 (2B4), CD160, GARP, OX40, CD137 (4-1BB), CD25,VISTA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6, CD39, CD73, CD4,CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20, CD23, CD24, CD38, CD93,IgM, B220 (CD45R), CD317, PD-L1, CD11b, Ly6G, ICAM-1, FAP, PDGFR,Podoplanin, and TIGIT.
 9. The method of claim 1, wherein the T cellreceptor complex-binding domain is a T cell receptor-binding domain or aCD3-binding domain.
 10. The method of claim 1, wherein the anticanceragent is an alkylating agent, an antimetabolite, a plant alkaloid, anantibiotic, a platinum compound, methylhydrazine, a kinase inhibitor, anenzyme, a histone deacetylase inhibitor, a retinoid, an antibody, or animmune checkpoint inhibitor.
 11. The method of claim 1, wherein theanticancer agent is a second antigen-binding molecule comprising: (1) acancer-specific antigen-binding domain; and (2) a tumor necrosis factor(TNF) superfamily-binding domain or tumor necrosis factor (TNF) receptorsuperfamily-binding domain.
 12. The method of claim 1, wherein theanticancer agent is a third antigen-binding molecule comprising: (1) acancer-specific antigen-binding domain; and (2) a T cell receptorcomplex-binding domain.
 13. The method of claim 12, wherein the secondantigen-binding molecule and/or the third antigen-binding moleculefurther comprises an FcRn-binding domain.
 14. The method of claim 14,wherein the FcRn-binding domain of the second antigen-binding moleculeand/or the third antigen-binding molecule is an antibody Fc regionhaving decreased Fcγ receptor-binding activity.
 15. The method of claim12, wherein the second antigen-binding molecule and/or the thirdantigen-binding molecule is a bispecific antibody.
 16. The method ofclaim 12, wherein the cancer-specific antigen-binding domain of thesecond antigen-binding molecule and/or the third antigen-bindingmolecule is a GPC3-binding domain.
 17. The method of claim 12, whereinthe TNF superfamily-binding domain or the TNF receptorsuperfamily-binding domain of the second antigen-binding molecule is aCD137-binding domain or a CD40-binding domain.
 18. The method of claim13, wherein the T cell receptor complex-binding domain of the thirdantigen-binding molecule is a T cell receptor-binding domain or aCD3-binding domain.
 19. The method of claim 1, wherein the cancer is anycancer selected from ovarian cancer, gastric cancer, esophageal cancer,pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma,breast cancer, malignant melanoma, non-small-cell lung cancer, cervicalcancer, glioblastoma, prostate cancer, neuroblastoma, chroniclymphocytic leukemia, papillary thyroid cancer, colorectal cancer, andB-cell non-Hodgkin's lymphoma.
 20. A method for enhancing effects ofinducing cytotoxicity, suppressing cell proliferation, inhibiting cellproliferation, activating an immune response, treating cancer, orpreventing cancer in an individual by a first antigen-binding molecule,each method comprising administering an effective amount of ananticancer agent to an individual, wherein the first antigen-bindingmolecule comprises: (1) a domain that binds to a molecule expressed onthe surface of a cell having an immune response-suppressing function;and (2) a T cell receptor complex-binding domain, and inhibits theimmune response-suppressing activity of the cell having an immuneresponse-suppressing function.
 21. A pharmaceutical composition fortreating or preventing cancer, the composition comprising a combinationof an anticancer agent and a first antigen-binding molecule thatcomprises: (1) a domain that binds to a molecule expressed on thesurface of a cell having an immune response-suppressing function; and(2) a T cell receptor complex-binding domain, and inhibits the immuneresponse-suppressing activity of the cell having an immuneresponse-suppressing function.
 22. The pharmaceutical composition ofclaim 21, which is a combination preparation.
 23. A kit comprising: (A)a pharmaceutical composition comprising a first antigen-binding moleculethat comprises: (1) a domain that binds to a molecule expressed on thesurface of a cell having an immune response-suppressing function; and(2) a T cell receptor complex-binding domain, and inhibits the immuneresponse-suppressing activity of the cell having an immuneresponse-suppressing function; (B) a container; and (C) an instructionor a label indicating that the first antigen-binding molecule and atleast one type of anticancer agent are administered in combination to anindividual for treating or preventing cancer in the individual.
 24. Amethod for inducing damage to a cancer cell or a cancer cell-comprisingtumor tissue, or a method for suppressing proliferation of a cancer cellor a cancer cell-comprising tumor tissue, by contacting a cell having animmune response-suppressing function and a cancer cell with ananticancer agent and a first antigen-binding molecule that comprises:(1) a domain that binds to a molecule expressed on the surface of thecell having an immune response-suppressing function; and (2) a T cellreceptor complex-binding domain, and inhibits the immuneresponse-suppressing activity of the cell having an immuneresponse-suppressing function.
 25. A method for enhancing effects ofinducing cytotoxicity, suppressing cell proliferation, inhibiting cellproliferation, activating immune response, treating cancer, orpreventing cancer in an individual by an anticancer agent, each methodcomprising administering to an individual an effective amount of a firstantigen-binding molecule that comprises: (1) a domain that binds to amolecule expressed on the surface of a cell having an immuneresponse-suppressing function; and (2) a T cell receptor complex-bindingdomain, and inhibits the immune response-suppressing activity of thecell having an immune response-suppressing function.
 26. The method ofclaim 13, wherein the second antigen-binding molecule and/or the thirdantigen-binding molecule further comprises an FcRn-binding domain. 27.The method of claim 13, wherein the second antigen-binding moleculeand/or the third antigen-binding molecule is a bispecific antibody. 28.The method of claim 13, wherein the cancer-specific antigen-bindingdomain of the second antigen-binding molecule and/or the thirdantigen-binding molecule is a GPC3-binding domain.